Discovery and characterization of orthosteric and allosteric muscarinic M2 acetylcholine receptor ligands by affinity selection-mass spectrometry.
Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify small-molecule hits from chemical libraries can provide a useful discovery approach that complements traditional assay systems. Affinity selection-mass spectrometry (AS-MS) is one such methodology that holds promise for providing selective and sensitive high-throughput screening platforms. Although AS-MS screening platforms have been used to discover small-molecule ligands of proteins from many target families, they have not yet been used routinely to screen integral membrane proteins. The authors present a proof-of-concept study using size exclusion chromatography coupled to AS-MS to perform a primary screen for small-molecule ligands of the purified muscarinic M2 acetylcholine receptor, a G-protein-coupled receptor. AS-MS is used to characterize the binding mechanisms of 2 newly discovered ligands. NGD-3350 is a novel M2-specific orthosteric antagonist of M2 function. NGD-3366 is an allosteric ligand with binding properties similar to the allosteric antagonist W-84, which decreases the dissociation rate of N-methyl-scopolamine from the M2 receptor. Binding properties of the ligands discerned from AS-MS assays agree with those from in vitro biochemical assays. The authors conclude that when used with appropriate small-molecule libraries, AS-MS may provide a useful high-throughput assay system for the discovery and characterization of all classes of integral membrane protein ligands, including allosteric modulators.
Whitehurst
Charles E
CE
NeoGenesis Pharmaceuticals, Inc., Cambridge, MA 02139, USA. charles.whitehurst@spcorp.com
Nazef
Naim
N
Annis
D Allen
DA
Hou
Yongmin
Y
Murphy
Denise M
DM
Spacciapoli
Peter
P
Yao
Zhiping
Z
Ziebell
Michael R
MR
Cheng
Cliff C
CC
Shipps
Gerald W
GW
Felsch
Jason S
JS
Lau
David
D
Nash
Huw M
HM
D000109:Acetylcholine; D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006168:Guinea Pigs; D007313:Insecta; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D018727:Muscarinic Antagonists / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects*; D011869:Radioligand Assay; D043585:Receptor, Muscarinic M2 / Q000737:chemistry* / Q000302:isolation & purification; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors*
D008024:Ligands; D018727:Muscarinic Antagonists; D043585:Receptor, Muscarinic M2; D043562:Receptors, G-Protein-Coupled; D000109:Acetylcholine