Fluorescence Correlation Spectroscopy in Drug Discovery: Study of Alexa532-Endothelin 1 Binding to the Endothelin ETAReceptor 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 ETAreceptor-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 ETAantagonist BQ-123, confirming the specific binding of Alexa532-ET1 to the endothelin ETAreceptor. 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.

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Lateral Mobility and Specific Binding to GABAAReceptors on Hippocampal Neurons Monitored by Fluorescence Correlation Spectroscopy†

Biochemistry ◽

10.1021/bi0263356 ◽

2003 ◽

Vol 42 (6) ◽

pp. 1667-1672 ◽

Cited By ~ 43

Author(s):

Oliver Meissner ◽

Hanns Häberlein

Keyword(s):

Fluorescence Correlation Spectroscopy ◽

Hippocampal Neurons ◽

Specific Binding ◽

Correlation Spectroscopy ◽

Lateral Mobility ◽

Fluorescence Correlation

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A fluorescence correlation spectroscopy study of ligand interaction with cytokinin-specific binding protein from mung bean

Biological Chemistry ◽

10.1515/bc.2010.005 ◽

2010 ◽

Vol 391 (1) ◽

Cited By ~ 9

Author(s):

Paweł Zawadzki ◽

Genowefa Ślósarek ◽

Jerzy Boryski ◽

Przemysław Wojtaszek

Keyword(s):

Ligand Binding ◽

Diffusion Coefficients ◽

Fluorescence Correlation Spectroscopy ◽

Mung Bean ◽

Specific Binding ◽

Binding Protein ◽

Correlation Spectroscopy ◽

Fluorescence Correlation ◽

Specific Binding Protein ◽

Two Populations

Abstract Cytokinins are essential plant hormones that regulate numerous physiological processes. Recently, a protein was identified in mung bean (Vigna radiata) and characterized as a cytokinin-specific binding protein (VrCSBP). Fluorescence correlation spectroscopy was used to investigate the interaction between VrCSBP and its ligands. The synthetic cytokinin, N-phenyl-N′-(4-pyridyl) urea, was labeled with two fluorophores, 7-nitro-2,1,3-benzoxadiazole and rhodamine B. Protein-ligand binding was analyzed in an equilibrium saturation binding experiment and confirmed by the competition assay. Surprisingly, it was found that VrCSBP binds not only to cytokinins, but also to gibberellins. In addition, in the presence of natural cytokinins and gibberellins, two populations of VrCSBP that differ in their diffusion coefficients were detected. The diffusion coefficients of these two populations could be related to mono- and dimeric states, which suggests a new mode of operation in ligand binding by VrCSBP, in which dimerization induced by natural ligands enhances the ligand binding capacity of the protein.

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