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Development of MS Binding Assays targeting the binding site of MB327 at the nicotinic acetylcholine receptor. 2018

S Sichler, and G Höfner, and S Rappenglück, and T Wein, and K V Niessen, and T Seeger, and F Worek, and H Thiermann, and F F Paintner, and K T Wanner
Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 Munich, Germany.

The bispyridinium compound MB327 has been shown previously to have a positive pharmacological effect against poisoning with organophosphorous compounds (OPCs). The mechanism by which it exerts its therapeutic effect seems to be directly mediated by the nicotinic acetylcholine receptor (nAChR). In the present study, the development of mass spectrometry based binding assays (MS Binding Assays) for characterization of the binding site of MB327 at the nAChR from Torpedo californica is described. MS Binding Assays follow the principle of radioligand binding assays, but do not, in contrast to the latter, require a radiolabeled reporter ligand, as the readout is in this case based on mass spectrometric detection. For [2H6]MB327, a deuterated MB327 analogue employed as reporter ligand in the MS Binding Assays, an LC-ESI-MS/MS method was established allowing for its fast and reliable quantification in samples resulting from binding experiments. Using centrifugation for separation of non-bound [2H6]MB327 from target-bound [2H6]MB327 in saturation and autocompetition experiments (employing native MB327 as competitor) enabled reliable determination of specific binding. In this way, the affinities for [2H6]MB327 (Kd=15.5±0.9μmolL-1) and for MB327 (Ki=18.3±2.6μmolL-1) towards the nAChR could be determined for the first time. The almost exactly matching affinities for MB327 and [2H6]MB327 obtained in the MS Binding Assays are in agreement with potencies previously found in functional studies. In summary, our results demonstrate that the established MS Binding Assays represent a promising tool for affinity determination of test compounds towards the binding site of MB327 at the nAChR.

UI MeSH Term Description Entries
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
D010622 Phencyclidine A hallucinogen formerly used as a veterinary anesthetic, and briefly as a general anesthetic for humans. Phencyclidine is similar to KETAMINE in structure and in many of its effects. Like ketamine, it can produce a dissociative state. It exerts its pharmacological action through inhibition of NMDA receptors (RECEPTORS, N-METHYL-D-ASPARTATE). As a drug of abuse, it is known as PCP and Angel Dust. 1-(1-Phenylcyclohexyl)piperidine,Angel Dust,CL-395,GP-121,Phencyclidine Hydrobromide,Phencyclidine Hydrochloride,Sernyl,Serylan,CL 395,CL395,Dust, Angel,GP 121,GP121
D011726 Pyridinium Compounds Derivatives of PYRIDINE containing a cation C5H5NH or radical C5H6N. Compounds, Pyridinium
D011869 Radioligand Assay Quantitative determination of receptor (binding) proteins in body fluids or tissue using radioactively labeled binding reagents (e.g., antibodies, intracellular receptors, plasma binders). Protein-Binding Radioassay,Radioreceptor Assay,Assay, Radioligand,Assay, Radioreceptor,Assays, Radioligand,Assays, Radioreceptor,Protein Binding Radioassay,Protein-Binding Radioassays,Radioassay, Protein-Binding,Radioassays, Protein-Binding,Radioligand Assays,Radioreceptor Assays
D011978 Receptors, Nicotinic One of the two major classes of cholinergic receptors. Nicotinic receptors were originally distinguished by their preference for NICOTINE over MUSCARINE. They are generally divided into muscle-type and neuronal-type (previously ganglionic) based on pharmacology, and subunit composition of the receptors. Nicotinic Acetylcholine Receptors,Nicotinic Receptors,Nicotinic Acetylcholine Receptor,Nicotinic Receptor,Acetylcholine Receptor, Nicotinic,Acetylcholine Receptors, Nicotinic,Receptor, Nicotinic,Receptor, Nicotinic Acetylcholine,Receptors, Nicotinic Acetylcholine
D002217 Carbachol A slowly hydrolyzed CHOLINERGIC AGONIST that acts at both MUSCARINIC RECEPTORS and NICOTINIC RECEPTORS. Carbamylcholine,Carbacholine,Carbamann,Carbamoylcholine,Carbastat,Carbocholine,Carboptic,Doryl,Isopto Carbachol,Jestryl,Miostat,Carbachol, Isopto
D002801 Cholinesterase Reactivators Drugs used to reverse the inactivation of cholinesterase caused by organophosphates or sulfonates. They are an important component of therapy in agricultural, industrial, and military poisonings by organophosphates and sulfonates. Insecticides, Organophosphate, Antagonists,Insecticides, Organothiophosphate, Antagonists,Organophosphate Insecticide Antagonists,Organothiophosphate Insecticide Antagonists,Antagonists, Organophosphate Insecticide,Antagonists, Organothiophosphate Insecticide,Insecticide Antagonists, Organophosphate,Insecticide Antagonists, Organothiophosphate,Reactivators, Cholinesterase
D002851 Chromatography, High Pressure Liquid Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed. Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D001665 Binding Sites The parts of a macromolecule that directly participate in its specific combination with another molecule. Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining

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