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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.
- Source :
-
Physical chemistry chemical physics : PCCP [Phys Chem Chem Phys] 2018 Nov 28; Vol. 20 (46), pp. 29212-29220. - Publication Year :
- 2018
-
Abstract
- 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, and addictions), neuroendocrinological alterations, and 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 on 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 the binding affinity of the new marker in the cellular environment.
- Subjects :
- Biomarkers chemistry
Fluorescent Dyes chemical synthesis
Fluorescent Dyes chemistry
Fluorescent Dyes pharmacology
Humans
Microscopy, Fluorescence
Molecular Structure
Receptors, Corticotropin-Releasing Hormone antagonists & inhibitors
Molecular Docking Simulation
Nanotechnology
Optical Imaging
Receptors, Corticotropin-Releasing Hormone chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 1463-9084
- Volume :
- 20
- Issue :
- 46
- Database :
- MEDLINE
- Journal :
- Physical chemistry chemical physics : PCCP
- Publication Type :
- Academic Journal
- Accession number :
- 30427333
- Full Text :
- https://doi.org/10.1039/c8cp06196c