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

Abstract

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. Copyright © 2012 Catherina Caballero-George et al.