| One of the difficulties in understanding the physiological role and connection to diseases and disorders of neuronal nicotinic acetylcholine receptors (nAChRs) lies in the distinction between the specific subtypes involved. It is of great importance to understand how specific nAChR subtypes are involved with normal physiology, diseases, and disorders. alpha4beta2* nAChRs have been implicated in a growing number of diseases and disorders; the most prominent being nicotine addiction. The studies presented in this dissertation characterize a novel, human alpha4beta2 (Halpha4beta2) selective antagonist as well as an allosteric site in which it binds. The primary contributions of this study are: (1) the discovery and characterization of the negative allosteric modulator (NAM), KAB-18, using calcium accumulation assays on recombinant Halpha4beta2 and Halpha3beta4 nAChRs, (2) the identification of the allosteric site in which KAB-18 binds using homology modeling, blind docking, and molecular dynamics (MD) simulations, (3) the validation of the allosteric site which was identified using computational modeling through the use of site-directed mutagenesis, (4) the construction of 3D models to predict the important binding interactions between this novel class of NAMs with Halpha4beta2 and Halpha3beta4 nAChRs, and (5) the identification of novel compounds that have similar "pharmacophores" as KAB-18 using ligand-based virtual screening. These studies provide much information regarding ligands that can interact selectively and potently with Halpha4beta2 nAChRs. These results also provide a novel mechanism for the negative allosterism of this class of NAMs on Halpha4beta2 and Halpha3beta4 nAChRs. The work presented herein provides a bridge to the design of new molecules from both a ligand based and structure based approach that preferentially bind and inhibit Halpha4beta2 nAChRs. This work will contribute to the field of nAChRs by providing a means to produce novel molecules; whether as molecular tools or as potential therapeutic drug candidates that target Halpha4beta2 nAChRs. |
