The overall goals of this proposed interdisciplinary effort continue to be characterization of benzodiazepine receptor (BDZR) pharmacology and design of behavioral selective ligands that act through these receptors. Design of these novel therapeutic agents will focus on two strategies: design of ligands that bind to the cerebellar """"""""Type I"""""""" BDZR and design of activity specific analogs. 1) the current 3D pharmacophore for the """"""""Type I"""""""" BDZR will be used to a) modify lead compounds that were discovered by searching 3D databases and b) design candidate """"""""Type I"""""""" selective antagonists by modification of selective agonists using our criteria for activation. The goal of further understanding of BDZR pharmacology will be addressed in three ways: a) determining if a novel alpidem-insensitive BDZR subtype identified in rat spinal cord membranes by determining if it is present in other brain regions and developing a 3D pharmacophore for recognition of this site; b) continuing to probe receptor heterogeneity by addressing the question of the number central BDZR subtypes in several additional brain regions using Fourier-derived affinity spectrum analysis used to successfully characterize receptor heterogeneity in spinal cord during the current grant period and validated in a known receptor system; c) determining the affinities at all sites identified in these different brain regions of the ligands that have been found in our current studies to display behavioral heterogeneity. 2) Design of activity specific analogs will be achieved in a multistep strategy based on identification of molecular determinants of recognition and activation at candidate receptors involved in mediation of each in vivo endpoint. These steps are: a) identification of receptors that mediate a particular in vivo endpoint as those to which compounds that are inactive at that endpoint do not bind with significant affinity; b) development of a 3D recognition pharmacophore for each candidate receptor by identification of calculated properties common to high affinity but absent in low affinity compounds; c) development of an activation pharmacophore for each receptor mediating a particular endpoint by identifying properties that are different among agonists, antagonists and inverse agonists; d) use of the 3D pharmacophores for recognition and activation relevant to each behavioral endpoint to search 3D databases for novel compounds that satisfy these criteria; e) acquisition or synthesis of selected compounds and evaluation for their ability to bind to BDZRs in different brain regions; evaluation of high affinity analogs at each of the five behavioral endpoints to determine to what extent their activity profiles match the predicted behavior and to identify activity selective analogs.
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