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 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.

Figure 1

Experimental setup for fluorescence correlation spectroscopy.

Figure 2

Comparison of autocorrelation curves of the Alexa532-ET1 (14.3 nM) binding to the ET_A receptor on living A10 cells. Control experiments (blue), coincubation with 1 μM BQ-123 (green) leads to an autocorrelation curve similar to that of the free ligand (red) indicating the displacement of the bound Alexa532-ET1.

Figure 3

Alexa532-ET1 binding to vascular smooth muscle cells. Averaged bound Alexa532-ET1 concentration versus the total Alexa532-ET1 concentration. The bound Alexa532-ET1 fraction was determined from the autocorrelation function for different Alexa532-ET1 concentrations (n = 5).

Figure 4

Results of the inositol phosphate (IP1) production assay after stimulation of A10 cells with different concentrations of nonlabeled endothelin 1 and Alexa532-ET1. Data are presented as the mean ± SEM of eight measurements.