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

Authors
  • Sichler, S1
  • Höfner, G1
  • Rappenglück, S1
  • Wein, T1
  • Niessen, K V2
  • Seeger, T2
  • Worek, F2
  • Thiermann, H2
  • Paintner, F F1
  • Wanner, K T3
  • 1 Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 Munich, Germany. , (Germany)
  • 2 Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstr. 11, 80937 Munich, Germany. , (Germany)
  • 3 Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 Munich, Germany. Electronic address: [email protected] , (Germany)
Type
Published Article
Journal
Toxicology letters
Publication Date
Nov 14, 2017
Identifiers
DOI: 10.1016/j.toxlet.2017.11.013
PMID: 29146291
Source
Medline
Keywords
License
Unknown

Abstract

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.

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