Discovery and Characterization of Orthosteric and Allosteric Muscarinic M2 Acetylcholine Receptor Ligands by Affinity Selection–Mass Spectrometry

Abstract

Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify smallmolecule 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 smallmolecule 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 M 2 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 M 2 -specific orthosteric antagonist of M 2 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 M 2 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. (Journal of Biomolecular

Discovery of a Novel Series of CHK1 Kinase Inhibitors with a Distinctive Hinge Binding Mode

“…12 ALIS has demonstrated its unique capability in screening label-free mixture-based libraries and finding hits that were subsequently developed into lead compounds in various classes of protein targets, including the anti-infective target Escherichia coli dihydrofolate reductase, 13 antibacterial AccC (acetyl coenzyme-A carboxylase), 14 HCV NS5B (hepatitis C virus nonstructural protein 5B) polymerase, 15−17 protein kinase CDK2 (cyclin-dependent kinase 2), 18 KSP (kinesin spindle protein) in oncology, 19 FABP4 (fatty acid binding protein-4) 20 and the lipid phosphatase SHIP2 (SH2 domain-containing inositol 5-phosphatase 2) 21 in diabetes, MK2 (mitogen-activated protein kinase-activated protein kinase 2) 22 and TACE (TNF-α converting enzyme) 23 in inflammation, and the GPCR (Gprotein-coupled receptor) muscarinic M 2 acetylcholine receptor. 24 Herein, we describe another example using ALIS to screen for novel inhibitors of the CHK1 protein and subsequent medicinal chemistry optimization.From the CHK1 ALIS screening of mixture-based libraries, compound 1 emerged as a high affinity ligand (ALIS K d <100 nM), and the series was selected for additional optimization. Biochemical assays confirmed its inhibitory activity against CHK1 with an IC 50 value of 75 nM with a clearly detectable incell activity (∼10 μM) that was hydroxyurea (HU)-dependent.…”

“…Interestingly, pronounced improvements in potency and selectivity against CDK2 were observed when the phenyl group was substituted with a para-phenoxy group (22). While para-NMe 2 substitution of phenyl group (23) resulted in a slight increase in potency, insertion of a methylene bridge between the dimethylamino and phenyl group (24) led to the complete loss of activity.…”

Application of high-throughput affinity-selection mass spectrometry for screening of chemical compound libraries in lead discovery

“…Recently, Schering-Plough scientists published their affinity-selection MS (AS-MS) method using a competitor ligand with a known affinity constant to displace a non-covalently target-bound compound from the screening library [3,4]. AS-MS affinity selection, even with a purified membrane receptor [4], was reported with an affinity ranking of binders. With this technology, the target is first incubated with the library compounds before separation of the target-binder complex from unbound substances by SEC.…”

“…Affinity selection discriminates between molecules that have an affinity to the biological target and those devoid of such an affinity. Binder information is the essential outcome of affinity-selection-based screening, although a ranking of the binders or even their characterization by affinity constants has been reported [2][3][4]. Mass spectrometry (MS) is preferentially used as the readout to identify binders by their molecular mass as this methodology can discriminate distinct chemical binders by their respective mass and has the tremendous benefit of very high detection sensitivity.…”

High-Throughput Affinity-Based Technologies for Small-Molecule Drug Discovery

“…Their activity was confirmed in a biochemical assay. 43 MS has also been used for direct identification of bound small-molecule compounds without a prior separation step. 44 It was achieved by detection of a complex of compounds and target protein.…”