Fluorescence Correlation Spectroscopy in Drug Discovery: Study of Alexa532-Endothelin 1 Binding to the Endothelin ETA Receptor to Describe the Pharmacological Profile of Natural Products

Fluorescence correlation spectroscopy and the newly synthesized Alexa532-ET1 were used to study the dynamics of the endothelin ETA receptor-ligand complex alone and under the influence of a semisynthetic selective antagonist and a fungal extract on living A10 cells. Dose-dependent increase of inositol phosphate production was seen for Alexa532-ET1, and its binding was reduced to 8% by the selective endothelin ETA antagonist BQ-123, confirming the specific binding of Alexa532-ET1 to the endothelin ETA receptor. Two different lateral mobilities of the receptor-ligand complexes within the cell membrane were found allowing the discrimination of different states for this complex. BQ-123 showed a strong binding affinity to the “inactive” receptor state characterized by the slow diffusion time constant. A similar effect was observed for the fungal extract, which completely displaced Alexa532-ET1 from its binding to the “inactive” receptor state. These findings suggest that both BQ-123 and the fungal extract act as inverse agonists

Impact of St. John’s wort extract Ze 117 on stress induced changes in the lipidome of PBMC

Abstract Background Membrane lipids have an important function in the brain as they not only provide a physical barrier segregating the inner and outer cellular environments, but are also involved in cell signaling. It has been shown that the lipid composition effects membrane fluidity which affects lateral mobility and activity of membrane-bound receptors. Methods Since changes in cellular membrane properties are considered to play an important role in the development of depression, the effect of St. John’s wort extract Ze 117 on plasma membrane fluidity in peripheral blood mononuclear cells (PBMC) was investigated using fluorescence anisotropy measurements. Changes in fatty acid residues in phospholipids after treatment of cortisol-stressed [1 μM] PBMCs with Ze 117 [10–50 µg/ml] were analyzed by mass spectrometry. Results Cortisol increased membrane fluidity significantly by 3%, co-treatment with Ze 117 [50 µg/ml] counteracted this by 4.6%. The increased membrane rigidity by Ze 117 in cortisol-stressed [1 μM] PBMC can be explained by a reduced average number of double bonds and shortened chain length of fatty acid residues in phospholipids, as shown by lipidomics experiments. Conclusion The increase in membrane rigidity after Ze 117 treatment and therefore the ability to normalize membrane structure points to a new mechanism of antidepressant action of the extract

The cientificWorldJOURNAL Research Article Fluorescence Correlation Spectroscopy in Drug Discovery: Study of Alexa532-Endothelin 1 Binding to the Endothelin ET A Receptor to Describe the Pharmacological Profile of Natural Products

Fluorescence correlation spectroscopy and the newly synthesized Alexa532-ET1 were used to study the dynamics of the endothelin ET A receptor-ligand complex alone and under the influence of a semisynthetic selective antagonist and a fungal extract on living A10 cells. Dose-dependent increase of inositol phosphate production was seen for Alexa532-ET1, and its binding was reduced to 8% by the selective endothelin ET A antagonist BQ-123, confirming the specific binding of Alexa532-ET1 to the endothelin ET A receptor. Two different lateral mobilities of the receptor-ligand complexes within the cell membrane were found allowing the discrimination of different states for this complex. BQ-123 showed a strong binding affinity to the "inactive" receptor state characterized by the slow diffusion time constant. A similar effect was observed for the fungal extract, which completely displaced Alexa532-ET1 from its binding to the "inactive" receptor state. These findings suggest that both BQ-123 and the fungal extract act as inverse agonists