MS Binding Assays with UNC0642 as reporter ligand for the MB327 binding site of the nicotinic acetylcholine receptor

Intoxications with organophosphorus compounds (OPCs) based chemical warfare agents and insecticides may result in a detrimental overstimulation of muscarinic and nicotinic acetylcholine receptors evolving into a cholinergic crisis leading to death due to respiratory failure. In the case of the nicotinic acetylcholine receptor (nAChR), overstimulation leads to a desensitization of the receptor, which cannot be pharmacologically treated so far. Still, compounds interacting with the MB327 binding site of the nAChR like the bispyridinium salt MB327 have been found to re-establish the functional activity of the desensitized receptor. Only recently, a series of quinazoline derivatives with UNC0642 as one of the most prominent representatives has been identified to address the MB327 binding site of the nAChR as well. In the present study, MS Binding Assays utilizing UNC0642 as a reporter ligand have been established. Thus, the binding of UNC0642 towards Torpedo-nAChR has been characterized in MS saturation and competition experiments. According to the results, UNC0642 addresses the MB327 binding site of the Torpedo-nAChR. This conclusion is further supported by the outcome of ex vivo studies performed with poisoned rat diaphragm muscles as well as by in silico studies predicting the binding mode of the most affine analog UNC0646 in the recently proposed binding site of MB327 (MB327-PAM-1). The new MS Binding Assays based on the commercially available reporter ligand UNC0642 as one of the most affine ligands for the MB327 binding site are a potent and valuable alternative to established assays.