Neuronal Nicotinic Acetylcholine Receptor (nAChR) is a ligand gated ion channel playing an important role in the cholinergic system. A broad range of various CNS drugs interact directly with the neurotransmitter binding sites or allosterically on several other binding domains of the receptor modulating its synaptic activity. Fast and reliable methods of measuring and characterization of this modulation is of great interest in CNS pharmacology and drug discovery.

New experimental and theoretical methods to characterize these interactions has been developed. Affinity chromatography technique, with the nAChR immobilized on the surface of the chromatographic stationary phase, can be used to describe the affinity of small ligands towards the receptor and the kinetics of this process. We used this method in fast screening of the series of constrained nicotine analogs and the method was able to successfully sort out the compounds with activity on nAChR. High agonistic activities were further confirmed in regular functional assays for these compounds.

Bioaffinity chromatography was also employed to determine the affinity of non-competitive (allosteric) inhibitors (NCIs) towards the receptor. This application is particularly important since other methods are hardly applicable to characterize the strength of binding for this class of ligands. The data collected for the series of non-competitive inhibitors was used to generate QSAR models describing the affinity of ligands towards the internal channel domain of nAChR and to predict functional inhibition in in vitro studies.

The molecular mechanisms of non-competitive inhibition were also elucidated with computational modeling techniques. The inner surface of the nAChR channel is regarded as the most common active site for binding and inhibition by allosteric NCIs. The molecular model of the channel domain was generated using homology modeling. Molecular docking procedure was employed and the series of active site - ligand molecular complexes were developed. The estimated values of free energy changes of binding (DG) obtained in these simulations were found to be well correlated with inhibitor affinities measured in chromatographic experiments.

The results of the computational simulations and QSAR modeling suggested the alternative mechanism of blocking action for the non-competitive inhibitors of ion channels. The tandem of chromatographic approach and computational modeling can be successfully employed for fast screening of pharmacological modulation of nAChRs by CNS drugs and new drug candidates. Various subtypes of the receptor are of increasing interest for medicinal chemists as potential drug targets and presented methods of ligand characterization can be used in drug discovery and design.

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