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

• Published in: Toxicology letters 2018-09, Vol.293, p.172-183
• Main Authors: Sichler, S., Höfner, G., Rappenglück, S., Wein, T., Niessen, K.V., Seeger, T., Worek, F., Thiermann, H., Paintner, F.F., Wanner, K.T.
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites - drug effects; Binding, Competitive - drug effects; Carbachol - metabolism; Cholinesterase Reactivators - pharmacology; Chromatography, High Pressure Liquid; LC–MS; Mass Spectrometry - methods; MB327; Models, Molecular; MS Binding Assays; nicotinic acetylcholine receptor; Phencyclidine - metabolism; Pyridinium Compounds - pharmacology; Radioligand Assay; Receptors, Nicotinic - drug effects; Reproducibility of Results; resensitizer; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Torpedo
• ISSN: 0378-4274; 1879-3169
• DOI: 10.1016/j.toxlet.2017.11.013

[Display omitted] •MS Binding Assays for MB327, a potential nAChR-resensitizer, were developed.For quantification of MB327 a fast and sensitive LC-ESI-MS/MS method was established and validated.•MB327 binding at the nAChR was characterized for the first time.•MB327 binding at the nAChR was characterized for the first time.•These MS Binding Assays are the first tool for development of structure affinity relationships at the MB327 binding site. 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.