scholarly journals ThermoFRET: A novel nanoscale G protein coupled receptor thermostability assay functional in crude solubilised membrane preparations

2020 ◽  
Author(s):  
David N Tippett ◽  
Brad Hoare ◽  
Tamara Miljus ◽  
David A Sykes ◽  
Dmitry B. Veprintsev
Keyword(s):  
Drug Discovery ◽  
Protein Purification ◽  
G Protein ◽  
High Throughput ◽  
Thermal Denaturation ◽  
Protein Unfolding ◽  
Orphan Receptor ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

AbstractSensitive protein stability assays for membrane proteins are crucial for developing purification protocols, for structural and biophysical characterisation and drug discovery. Here, we describe a novel high-throughput 384-well FRET-based thermostability methodology, ThermoFRET, allowing for the ultrasensitive determination of G protein coupled receptor (GPCR) stability. This method measures FRET between a terbium-cryptate labelled GPCR and BODIPY-FL-Cystine, a thiolreactive dye that reacts with cysteine residues exposed upon protein unfolding in response to thermal denaturation. ThermoFRET is functional in crude solubilised membrane preparations, without protein purification and can detect receptor stabilising ligands, making it ideally suited for orphan receptor screening.

scholarly journals Cutting Edge: Identification of the Orphan Receptor G-Protein-Coupled Receptor 2 as CCR10, a Specific Receptor for the Chemokine ESkine

2000 ◽  
Vol 164 (7) ◽  
pp. 3460-3464 ◽  
Author(s):  
David I. Jarmin ◽  
Miriam Rits ◽  
Dalena Bota ◽  
Norma P. Gerard ◽  
Gerard J. Graham ◽  
...  
Keyword(s):  
G Protein ◽  
Cutting Edge ◽  
Orphan Receptor ◽  
Specific Receptor ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals High-Throughput Fluorescence Polarization Assay to Identify Ligands Using Purified G Protein-Coupled Receptor

2019 ◽  
Vol 24 (9) ◽  
pp. 915-927
Author(s):  
P. Heine ◽  
G. Witt ◽  
A. Gilardi ◽  
P. Gribbon ◽  
L. Kummer ◽  
...  
Keyword(s):  
G Protein ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
Binding Pocket ◽  
G Protein Coupled Receptor ◽  
Library Screen ◽  
A Cell ◽  
Fluorescence Polarization Assay ◽  
G Protein Coupled

The development of cell-free high-throughput (HT) methods to screen and select novel lead compounds remains one of the key challenges in G protein-coupled receptor (GPCR) drug discovery. Mutational approaches have allowed the stabilization of GPCRs in a purified and ligand-free state. The increased intramolecular stability overcomes two major drawbacks for usage in in vitro screening, the low receptor density on cells and the low stability in micelles. Here, an HT fluorescence polarization (FP) assay for the neurotensin receptor type 1 (NTS1) was developed. The assay operates in a 384-well format and is tolerant to DMSO. From a library screen of 1272 compounds, 12 (~1%) were identified as primary hits. These compounds were validated in orthogonal assay formats using surface plasmon resonance (SPR), which confirmed binding of seven compounds (0.6%). One of these compounds showed a clear preference for the orthosteric binding pocket with submicromolar affinity. A second compound revealed binding at a nonorthosteric binding region and showed specific biological activity on NTS1-expressing cells. A search of analogs led to further enhancement of affinity, but at the expense of activity. The identification of GPCR ligands in a cell-free assay should allow the expansion of GPCR pharmaceuticals with antagonistic or agonistic activity.

scholarly journals The Significance of G Protein-Coupled Receptor Crystallography for Drug Discovery

2011 ◽  
Vol 63 (4) ◽  
pp. 901-937 ◽  
Author(s):  
John A. Salon ◽  
David T. Lodowski ◽  
Krzysztof Palczewski
Keyword(s):  
Drug Discovery ◽  
G Protein ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

G Protein-Coupled Receptor Transmembrane Binding Pockets and their Applications in GPCR Research and Drug Discovery: A Survey

2011 ◽  
Vol 11 (15) ◽  
pp. 1902-1924 ◽  
Author(s):  
Nicole A. Kratochwil ◽  
Silvia Gatti-McArthur ◽  
Marius C. Hoener ◽  
Lothar Lindemann ◽  
Andreas D. Christ ◽  
...  
Keyword(s):  
Drug Discovery ◽  
G Protein ◽  
G Protein Coupled Receptor ◽  
Binding Pockets ◽  
G Protein Coupled

scholarly journals Identification of the first surrogate agonists for the G protein-coupled receptor GPR132

RSC Advances ◽  
2015 ◽  
Vol 5 (60) ◽  
pp. 48551-48557 ◽  
Author(s):  
Mohamed A. Shehata ◽  
Hanna Belcik Christensen ◽  
Vignir Isberg ◽  
Daniel Sejer Pedersen ◽  
Andreas Bender ◽  
...  
Keyword(s):  
G Protein ◽  
Binding Mode ◽  
Orphan Receptor ◽  
G Protein Coupled Receptor ◽  
Structure Activity Relationships ◽  
Structure Activity ◽  
G Protein Coupled

We report the first pharmacological tool agonist for in vitro characterization of the orphan receptor GPR132, preliminary structure–activity relationships based on 32 analogs and a suggested binding mode from docking.

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