scholarly journals High-throughput identification of G protein-coupled receptor modulators through affinity mass spectrometry screening

2018 ◽  
Vol 9 (12) ◽  
pp. 3192-3199 ◽  
Author(s):  
Shanshan Qin ◽  
Mengmeng Meng ◽  
Dehua Yang ◽  
Wenwen Bai ◽  
Yan Lu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
G Protein ◽  
High Throughput ◽  
G Protein Coupled Receptor ◽  
Receptor Modulators ◽  
G Protein Coupled

High-throughput identification of GPCR modulators through affinity MS screening.

Analysis of an Intact G-Protein Coupled Receptor by MALDI-TOF Mass Spectrometry:  Molecular Heterogeneity of the Tachykinin NK-1 Receptor

2007 ◽  
Vol 79 (6) ◽  
pp. 2189-2198 ◽  
Author(s):  
Isabel D. Alves ◽  
Emmanuelle Sachon ◽  
Gerard Bolbach ◽  
Lynda Millstine ◽  
Solange Lavielle ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
G Protein ◽  
Molecular Heterogeneity ◽  
G Protein Coupled Receptor ◽  
Maldi Tof Mass Spectrometry ◽  
Maldi Tof ◽  
G Protein Coupled

Characterization of G-Protein Coupled Receptor Modulators Using Homogeneous cAMP Assays

2012 ◽  
pp. 171-180 ◽  
Author(s):  
Daniel L. Bassoni ◽  
Qumber Jafri ◽  
Sunitha Sastry ◽  
Mahesh Mathrubutham ◽  
Tom S. Wehrman
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptor ◽  
Receptor Modulators ◽  
G Protein Coupled

scholarly journals Accelerating the Throughput of Affinity Mass Spectrometry-Based Ligand Screening toward a G Protein-Coupled Receptor

2019 ◽  
Vol 91 (13) ◽  
pp. 8162-8169 ◽  
Author(s):  
Yan Lu ◽  
Shanshan Qin ◽  
Bingjie Zhang ◽  
Antao Dai ◽  
Xiaoqing Cai ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
G Protein ◽  
G Protein Coupled Receptor ◽  
Ligand Screening ◽  
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 A Novel High Throughput Chemiluminescent Assay for the Measurement of Cellular Cyclic Adenosine Monophosphate Levels

2000 ◽  
Vol 5 (4) ◽  
pp. 239-247 ◽  
Author(s):  
Anthony C. Chiulli ◽  
Karen Trompeter ◽  
Michelle Palmer
Keyword(s):  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Receptor Activation ◽  
G Protein Coupled Receptor ◽  
Wide Range ◽  
G Protein Coupled ◽  
Camp Levels

The second messenger 3′, 5′-cyclic AMP (cAMP) is a highly regulated molecule that is governed by G protein-coupled receptor activation and other cellular processes. Measurement of cAMP levels in cells is widely used as an indicator of receptor function in drug discovery applications. We have developed a nonradioactive ELISA for the accurate quantitation of cAMP levels produced in cell-based assays. This novel competitive assay utilizes chemiluminescent detection that affords both a sensitivity and a dynamic assay range that have not been previously reported with any other assay methodologies. The assay has been automated in 96- and 384-well formats, providing assay data that are equivalent to, if not better than, data generated by hand. This report demonstrates the application of this novel assay technology to the functional analysis of a specific G protein-coupled receptor, neuropeptide receptor Y1, on SK-N-MC cells. Our data indicate the feasibility of utilizing this assay methodology for monitoring cAMP levels in a wide range of functional cell-based assays for high throughput screening.

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