The objective of this proposal is to define the interaction between an enzyme and a highly specific competitive inhibitor. The enzyme being studied is a mammalian cAMP-specific isozyme of phosphodiesterase (PDE) that is strongly implicated in learning and memory functions. Inhibitors of this enzyme are pharmacologically useful in the treatment of some human pathologies. The work involves the use of a novel in vivo drug-resistance selection for mutants of a mammalian enzyme expressed in yeast. This reconstitution of a mammalian gene product into yeast cells results in a simplified system and a mechanism for the isolation of rare drug resistant mutants. The selection to be used is designed to isolate mammalian PDE mutants that are incapable of interacting with a competitive inhibitor (rolipram), but are not affected in substrate (cAMP) binding or hydrolysis. This work should lead to a better understanding of the nature of the interaction between a mammalian PDE enzyme and a pharmacologically useful drug. In addition, the methods employed in this research should prove useful in both the design and testing of more potent inhibitors of this enzyme. The methodology will also be extended to the study of other mammalian enzymes. These new techniques could provide an invaluable tool for the analysis of a wide variety of inhibitor-enzyme interactions.
