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Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample's impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GP CR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology(1,2). The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y-1, Y-2, Y-4 and Y-5 receptors, with different affinity and selectivity(3). NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y-1 receptor (Y1R)(4). A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity(4), tumour(1) and bone loss(5). However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability(6). Here we report crystal structures of the human Y1R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 angstrom resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y1R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery that targets NPY receptors.

Label-free, holistic assays, monitoring, for example, the impedance of cells on electrodes, are gaining increasing popularity in the evaluation of G-protein-coupled receptor (GPCR) ligands. It is the strength of these approaches to provide the integrated cellular response non-invasively, highly automated and with a device-dependent time resolution down to several milliseconds. With an increasing number of samples to be studied in parallel, the available time resolution is, however, reduced and the cost for the disposable sensor arrays may become limiting. Inspired by protocols from organ pharmacology, we investigated a simple serial agonist addition assay that circumvents these limitations in impedance-based cellular assays. Using a serial addition of increasing concentrations of a GPCR agonist while continuously monitoring the sample's impedance, we were able to establish a full concentration-response curve for the endogenous agonist histamine on a single layer of U-373 MG cells endogenously expressing the histamine 1 receptor (H1R). This approach is validated with respect to conventional, parallel agonist addition protocols and studies using H1R antagonists such as mepyramine. Applicability of the serial agonist addition assay was shown for other GPCRs known for their signaling via one of the canonical G-protein pathways, Gq, Gi/0 or Gs as well. The serial agonist addition protocol has the potential to further strengthen the output of label-free analysis of GPCR activation.

We describe the synthesis of spherical poly(vinyl butyral) (PVB) nanobeads by controlled precipitation via addition of non-solvent. Effects of various reaction parameters on nanoparticle size were investigated by dynamic light scattering and electron microscopy. The ability to incorporate dopant molecules was studied using a fluorescent perylene derivative as a model additive, and the dye-doped nanoparticles were investigated by confocal microscopy. In an optimized experimental protocol, PVB nanoparticles were obtained that were efficiently taken up by human cancer cells devoid of coating. The novel nanospheres are economic, easy to prepare and capable of incorporating additives. Lacking cytotoxicity in vitro, PVB nanobeads are attractive with respect to various potential applications such as optical imaging and particle tracking, diagnostics, and drug delivery.

Estrogen and iron play critical roles in a female body development and were investigated in the present study in relation to in vitro cell proliferation. Prempro, a hormone replacement therapy drug, and 17beta-estradiol (E2) were shown to increase cell proliferations in estrogen receptor positive (ER+) cells independent of progesterone receptor (PR) status. For example, increased cell proliferation was observed in ER+/PR+ human breast cancer MCF-7, its matching non-cancerous human breast epithelial MCF-12A, and ER+/PR+ murine mammary cancer MXT+ cells, but not in ER-/PR- MDA-MB-231, its matching non-cancerous MCF-10A, and MXT- (ER-/PR+) cells. By mimicking post-menopausal conditions of high estrogen in local breast tissue and increased iron levels due to cessation of menstrual periods, E2 and iron were shown to exert synergistic effects on proliferation of MCF-7 cells and significantly increased Ki67 and proliferating cell nuclear antigen. Western blotting of E2-treated ER+ but not ER- cells showed that E2 also increased transferrin receptor (TfR). Further studies are needed to assess the mitogenic effects of iron and estrogen in normal post-menopausal breast.

The histamine (HA) receptor subtype 1 (H1R) and H4R are expressed on immune cells and contribute to an inflammatory reaction. Both receptor subtypes individually enhance the intracellular concentrations of calcium and regulate the accumulation of cAMP, increase MAPK activity, and regulate expression of e.g., inflammatory genes. In a previous study we characterized and compared signaling pathways of the murine orthologs of the H1R and the H4R recombinantly expressed at comparable levels in HEK 293 cells. In the present study, we aimed at analyzing possible interactions of the signaling pathways emerging at the mH1R and the mH4R. Therefore, we co-expressed both receptor subtypes at comparable levels in HEK 293 cells and investigated HA-induced signaling parameters such as the concentrations of intracellular calcium and cAMP, phosphorylation of the MAPKs p38, ERK 1, and ERK 2, and of the transcription factor CREB, and expression of the immediate early gene EGR-1. We demonstrate that the intracellular concentrations of calcium and cAMP as well as the EGR-1 expression are regulated exclusively via the mH1R. In contrast, both receptor subtypes H1R and H4R synergize in HA-induced MAPK activation. This synergism most probably relies on signaling pathways independent of the second messenger calcium and cAMP. In summary, we provide evidence that the mH1R inhibits or dampens the function of the co-expressed mH4R regarding specific parameters, while other signaling events are regulated cooperatively by the mH1R and the mH4R.

The preparation of cyclic 10-membered tetrahydroxy enediynes is reported. The synthesis starts from tartaric acid and allows the control of the relative stereochemistry. Acetal protection of the 2,3-hydroxy groups stabilizes the enediyne during synthesis. Removal of the cyclic protecting group with EtSH/TFA transforms the stable compounds into reactive enediynes, and the rate constants of their cyclization were determined in benzene and water. The cytoxicity of the activated compounds was assayed against tumor cells in vitro, but the growth inhibitory effect was weak compared to cisplatin.

The water-soluble porphyrin-platinum complex diammine[7,12-bis[1-(polyethyleneglycol-750-monomethylether-1-yl)ethyl]-3,8,13,17-tetramethylporphyrin-2,18-dipropionato]platinum(II) (PEG-HPPt) was studied with respect to cellular accumulation, subcellular localization, behavior in 3D-cell aggregates and degree of DNA platination on the low-differentiated J82 cells, a model of invasive bladder cancer, and UROtsa, a normal urothelial cell line. Accumulation studies with 2D and spheroid cell cultures revealed that the concentration of PEG-HPPt was 1.7-times higher in J82 cancer cells than in UROtsa cells. Despite its high molecular weight, penetration of PEG-HPPt was not restricted to the peripheral cells of the spheroids. Fluorescence microscopic analysis showed that PEG-HPPt was localized in essential cellular targets of photodynamic therapy. DNA platination in J82 and UROtsa cells was higher by PEG-HPPt than by cisplatin, whereas there was no significant difference between the two cell lines.

Four porphyrin-platinum complexes, conceived as a new approach in cancer therapy by combining the cytostatic activity of cisplatin or oxaliplatin and the photodynamic effect of hematoporphyrin in the same molecule, were studied in detail with respect to solubility and stability in cell culture medium as well as in terms of cytotoxicity and phototoxicity against J82 bladder cancer cells and UROtsa, normal urothelial cells. This study demonstrated that the most active and promising compound among the porphyrin-platinum conjugates investigated was the water-soluble porphyrin-platinum complex 4 (diammine[7,12-bis[1-(polyethyleneglycol-750-monomethylether-1-yl)ethyl]-3,8,13,17-tetramethylporphyrin-2,18-dipropionato]platinum(II)) which exhibited a synergistic antiproliferative effect compared to cisplatin and hematoporphyrin alone or a combination of the drugs.

K[PtCl3(NH3)] reacts with an equimolar amt. of 3-(2-amino-ethoxy)estrone (an) or -estradiol (ad) to give cis-[PtCl2(NH3)L] (L = an, ad). The reaction of cis-PtCl2(NH3)2 with the amino L leads to cis-[PtCl(NH3)2L]Cl (I). I (L = ad) is active against the hormone sensitive human breast cancer cell line MCF-7 and L 1210 of the mouse., CAN 115:196743 78-7 Inorganic Chemicals and Reactions 136392-25-1P Role: SPN (Synthetic preparation), FORM (Formation, nonpreparative), PREP (Preparation) (formation of, in prepn. of mono- and diammine analogs); 136392-21-7P; 136392-22-8P; 136392-23-9P Role: BAC (Biological activity or effector, except adverse), BSU (Biological study, unclassified), SPN (Synthetic preparation), THU (Therapeutic use), BIOL (Biological study), PREP (Preparation), USES (Uses) (prepn. and antitumor activity of); 136392-24-0P; 136458-88-3P; 136521-64-7P Role: SPN (Synthetic preparation), PREP (Preparation) (prepn. of)