There continues to be a need for effective yet safe treatments for anxiety and related disorders. Current treatments such as the benzodiazepines which target many of the gamma-aminobutyric acid receptor (GABA-A receptor) subtypes are effective, but patients suffer from a number of side-effects. The identification of compounds targeting only the anxiolytic alpha-2 GABA-A receptor subtype has been hampered by a lack of structure/function information needed for effective lead optimization and drug discovery. Phase I of this project will use site-directed mutagenesis of unnatural amino acids to delineate the structure-functions relationships responsible for binding to the alpha-2 GABA-A receptor subtype. The phase I goals are: ? The use of the nonsense suppression method for incorporating unnatural amino acids into specific positions in the (alpha-1)2(beta-3)2(gamma-2) and (alpha-2)2(beta-3)2(gamma-2) GABA-A receptor subtypes. This involves site-directed mutagenesis studies in which unnatural amino acids will be incorporated at specific positions on the alpha and gamma subunits of GABA-A receptor. Validation of the mutated proteins will be using electrophysiological recording techniques. ? Assess the activity of the natural ligand GABA and five structurally diverse allosteric modulators of GABA-A receptor against each of the mutated receptors and develop a Receptophore TM model of alpha-2 receptor subtype selectivity. Phase II will use the information generated during Phase I to design and synthesize alpha-2 receptor subtype selective GABA-A receptor compounds. Further lead optimization, pharmacology and toxicology will provide safer therapies for the treatment of anxiety disorders. ? ? ?
