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  • Very recently Niessen et al have described the resensitizing

    2022-11-17

    Very recently, Niessen et al. have described the resensitizing effect of MB327 on nAChR performing solid-supported membrane (SSM)-based electrophysiological experiments (Niessen et al., 2016) using plasma membrane preparations obtained from Torpedo californica. These findings validated and underlined previous results of Seeger et al. who showed the restoration of muscular activity of soman-impaired rat diaphragm and human intercostal muscle preparations (Seeger et al., 2012). Before that, in vivo experiments have indicated that MB327 is able to restore neuromuscular transmission of guinea-pig diaphragms, improving survival in sarin, soman, and tabun poisoned guinea-pigs (Turner et al., 2011). In addition, it was demonstrated that MB327 is not binding to the TH287 receptor binding site (Niessen et al., 2011, Niessen et al., 2013) which is referred to as orthosteric binding site of the nAChR. These findings indicate that MB327 is able to revert the desensitized conformation of nAChR into the active form, but the mode of action and specific binding sites of MB327 and related compounds are still unknown. Taking MB327 as starting point for the development of new more potent compounds against OP poisoning, it would be of great benefit to have a detailed knowledge on the binding site of MB327. This would enable a detailed understanding of the interactions of MB327 with nAChR by inspection of the putative binding poses. Structure based molecular design methods like docking or de novo ligand design (Klebe and Böhm, 1996) could lead to compounds with new structures with enhanced binding affinity and an improved pharmacologic effect. The only 3D structure showing the complete nAChR including extracellular and transmembrane domains is the 3D structure of Torpedo marmorata published by N. Unwin in 2005 (Unwin, 2005). It has a resolution of only 4Å and shows the closed conformation of the receptor that had been crystallized in the absence of acetylcholine. In order to identify putative binding sites of MB327 at this nAChR 3D structure that serves as a substitute for the unknown 3D structures of human nAChR as mentioned above, we have searched the complete surface and accessible possible binding cavities inside and outside the channel pore. To this end, we have used the docking software GOLD (Verdonk and Cole, 2003), the nAChR 3D structure as target, and MB327 as ligand. Known agonists like acetylcholine, carbachol, epibatidine, and nicotine (Fig. 1) were included as ligands in the docking calculations to assure the validity of the method that had to find the orthosteric binding sites for these ligands. The search area used for the docking calculations could not be set to include the complete nAChR in one docking run because of program limitations. Hence, it was divided into eight smaller overlapping parts which are described in detail in the Materials and Methods section.
    Materials and methods The 3D structures of the investigated ligands in Fig. 1 including the correct protonations were generated with ChemAxon Marvin Sketch and molconvert software (Chemaxon, 2015). In the case of the nitrogen atom in nicotine the protonation leads to two stereoisomers which were both used for docking calculations and only the best docking scores are reported. The protein structure of the nAChR of Torpedo marmorata with the PDBID 2bg9 was taken directly from the Protein Data Bank (Berman et al., 2000). Forcefield parameters, protonation and partial charges were assigned in Accelrys Discovery Studio (Accelrys Software Inc, 2014). No further refinement or energy minimization of the 3D structure was performed. The dockings with GOLD software were prepared and started using the Discovery Studio Client 4.1 from Accelrys. GOLD version 5.2.2 was used. The complete nAChR protein could not be used as a search area because of program limitations. Due to that, the docking calculations have been split in smaller parts by limiting the sizes of the corresponding search spheres used (Fig. 2). In total eight different search spheres have been set up of which five spheres covering the outside surface of the extracellular domain of the nAChR (spheres 1, 2, 3, 4, and 5 in Table 1) and three spheres are located inside the channel pore (spheres 6, 7, and 8 in Table 1). The labeling of the search sites corresponds to the names given in Table 1. All x-, y- and z-coordinates given are related to the electron microscopy structure of nAChR (PDBID 2bg9) used in this study.