Planta Med 2008; 74 - PG37
DOI: 10.1055/s-0028-1084790

Fluorescence Correlation Spectroscopy (FCS) a powerful biophysic method for drug discovery: study of Alexa532-endothelin 1 binding on the endothelin ETA receptor on living cells

C Caballero-George 1, 3, T Sorkalla 2, E Bermingham 3, H Häberlein 2
  • 1INDICASAT-AIP, P.O. Box 0816–02852, Panama
  • 2Institut für Physiologische Chemie, Nussallee, Bonn 53115, Germany
  • 3Smithsonian Tropical Research Institute, P.O. Box 0843–03092, Panama

FCS is a powerful new method for examining molecular interactions on living cells, at real time without disturbing the ligand-receptor interaction [1]. The search for selective endothelin ETA (ETA) receptor antagonists is a top priority in the discovery of drugs to treat cardiovascular disorders. To investigate ETA receptor binding studies by FCS, the activated fluorophore Alexa Fluor 532 succinimidyl ester was coupled to endothelin 1 (ET1) to produce the ligand Alexa532-ET1. Monolabeled Alexa532-ET1 was purified by SEC and identified by MALDI-TOF experiments (m/z 3099, [M-H]). Binding experiments were carried out on vascular smooth muscle cells (A10) in Locke's solution at 20°C. The diffusion time (τ) of free Alexa532-ET1 was 92.2±1.9 (n=9)µs (D free =108.4±2.3µm2/s). In equilibrium, 25% of 5 nM Alexa532-ET1 was bound to the receptor. Two different lateral mobilities of the receptor-ligand complex within the cell membrane were found τ1 =0.8±0.1 and τ2=49.9±0.1 (n=13)ms (D bound1=12.5±4.0 and D bound2=0.2±0.1µm2/s) allowing the discrimination of different states for the receptor-ligand complex. In the same line, the binding of 5 nM Alexa532-ET1 was inhibited by the selective ETA antagonist BQ-123 (only 5% of non-specific binding was measured) confirming the presence of ETA receptors on A10 cells. The major advantage of FCS over other ligand-binding techniques like radioreceptor assays, is the characterization of the dynamics of receptor-ligand complexes in the biomembrane of living cells in real time at very dilute concentrations. FCS offers a state-of-the-art tool for drug discovery of natural products and the evaluation of their chemical kinetics.

Acknowledgements: National Secretary of Science and Technology (SENACYT), grant COL06–006 from the Panamanian government.

References: 1. Vukojević, V. et al. (2005) Cell Mol Life Sci 62: 535–550.