A fluorescence nanoscopy marker for Corticotropin-Releasing Hormone type 1 receptor: computer design, synthesis, signaling effects, super-resolved fluorescence imaging, and in-situ affinity constant in cells.

NATALIA G. ARMANDO*; LUCIANA GIORDANO; SUSANA SILBERSTEIN*; ALAN M. SZALAI*; FERNANDO D. STEFANI; CLAUDIO N. CAVASOTTO; FEDERICO M. BARABAS; SARA E. BARI; PEDRO F. ARAMENDÍA

PHYSICAL CHEMISTRY CHEMICAL PHYSICS

ROYAL SOC CHEMISTRY

Lugar: CAMBRIDGE; Año: 2018 p. 29212 - 29220

1463-9076

Class B G protein-coupled receptors (GPCRs) are involved in a variety of human pathophysiological states. These groups of membrane receptors are less studied than class A GPCRs due to the lack of structural information, delayed small molecule drug discovery, and scarce fluorescence detection tools available. The class B Corticotropin-Releasing Hormone type 1 Receptor (CRHR1) is a key player in the stress response whose dysregulation is critically involved in stress-related disorders: psychiatric conditions (i.e. depression, anxiety, addictions), neuroendocrinological alterations, neurodegenerative diseases. Here, we present a strategy to label GPCRs with a small fluorescent antagonist that permits the observation of the receptor in live cells through stochastic optical reconstruction microscopy (STORM) with 23 nm resolution. The marker, an aza-BODIPY derivative, was designed based on computational docking studies, then synthesized, and finally tested in biological cells. Experiments in hippocampal neurons demonstrate antagonist effects in similar concentrations as the well-established antagonist CP-376395. A quantitative analysis of two color STORM images enabled the determination of binding affinity of the new marker in the cellular environment.