stepwise-dosing-protocol-for-increased-throughput-label-free

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

University of Regensburg

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.

Combined chemotherapeutic and photodynamic treatment on human bladder cells by hematoporphyrin-platinum(II) conjugates.

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.

Distribution and subcellular localization of a water-soluble hematoporphyrin-platinum(II) complex in human bladder cancer cells.

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. 

Histamine H(1)- and H(4)-receptor signaling cooperatively regulate MAPK activation.

ABCG2 is an ATP-binding cassette (ABC) transporter that protects tissues against xenobiotics, affects the pharmacokinetics of drugs and contributes to multidrug resistance. Although many inhibitors and modulators of ABCG2 have been developed, understanding their structure-activity relationship requires high-resolution structural insight. Here, we present cryo-EM structures of human ABCG2 bound to synthetic derivatives of the fumitremorgin C-related inhibitor Ko143 or the multidrug resistance modulator tariquidar. Both compounds are bound to the central, inward-facing cavity of ABCG2, blocking access for substrates and preventing conformational changes required for ATP hydrolysis. The high resolutions allowed for de novo building of the entire transporter and also revealed tightly bound phospholipids and cholesterol interacting with the lipid-exposed surface of the transmembrane domains (TMDs). Extensive chemical modifications of the Ko143 scaffold combined with in vitro functional analyses revealed the details of ABCG2 interactions with this compound family and provide a basis for the design of novel inhibitors and modulators.

Structural basis of small-molecule inhibition of human multidrug transporter ABCG2.

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology  . 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, Y, Y and Y receptors, with different affinity and selectivity  . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y receptor (YR)  . A number of peptides and small-molecule compounds have been characterized as YR antagonists and have shown clinical potential in the treatment of obesity  , tumour  and bone loss  . However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability  . Here we report crystal structures of the human YR bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of YR to several structurally diverse antagonists and the determinants of ligand selectivity. The YR 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 YR can enable structure-based drug discovery that targets NPY receptors.

Article	Year
Combined chemotherapeutic and photodynamic treatment on human bladder cells by hematoporphyrin-platinum(II) conjugates. Cancer letters\| PubMed ID: 14732225	2004
Distribution and subcellular localization of a water-soluble hematoporphyrin-platinum(II) complex in human bladder cancer cells. Cancer letters\| PubMed ID: 15488635	2004
Histamine H(1)- and H(4)-receptor signaling cooperatively regulate MAPK activation. Biochemical pharmacology\| PubMed ID: 26385311	2015
Structural basis of small-molecule inhibition of human multidrug transporter ABCG2. Nature structural & molecular biology\| PubMed ID: 29610494	2018
Structural basis of ligand binding modes at the neuropeptide Y Y receptor. Nature\| PubMed ID: 29670288	2018

Guenther Bernhardt

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