Fluorescence Correlation Spectroscopy in Drug Discovery: Study of Alexa532-Endothelin 1 Binding to the Endothelin ET_AReceptor 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.

Modeling craniofacial development reveals spatiotemporal constraints on robust patterning of the mandibular arch

“…We model the two gradients as time-independent, exponentially declining functions from ventral to dorsal, solving the steady state with a source term at the ventral domain boundary and constant morphogen degradation. Measurements of diffusion coefficients for Edn1 range from 104.2–140.5 μm 2 /s [ 51 , 52 ], and for Bmps range from 2.0–4.4 μm 2 /s [ 53 , 54 ], suggesting that Edn1 diffuses farther dorsally than Bmps. Grem2, a Bmp antagonist expressed dorsally, also acts to restrict Bmp signalling to the ventral arch [ 30 ].…”

Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells

“…Techniques like FCS helped to define structure-function relationships among plasma membrane receptors (Caballero-George et al, 2012;Owen et al, 2009;Sieben, Kaminski, Kubitscheck, & Häberlein, 2011;Suzuki, Ritchie, Kajikawa, Fujiwara, & Kusumi, 2005). For example, epinephrine was found to induce dynamic regulations of the β 2adrenergic receptor exhibiting very rapid lateral diffusion (288 × 10 −10 cm 2 /s) for several minutes before reducing its mobility to 10 × 10 −10 cm 2 /s.…”

Recent applications of fluorescence correlation spectroscopy in live systems

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