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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS031911-02
Application #
2269863
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1993-08-01
Project End
1996-07-31
Budget Start
1994-08-01
Budget End
1995-07-31
Support Year
2
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Biochemistry
Type
Organized Research Units
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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Atienza, J M; Susanto, D; Huang, C et al. (1999) Identification of inhibitor specificity determinants in a mammalian phosphodiesterase. J Biol Chem 274:4839-47
Pillai, R; Staub, S F; Colicelli, J (1994) Mutational mapping of kinetic and pharmacological properties of a human cardiac cAMP phosphodiesterase. J Biol Chem 269:30676-81
Pillai, R; Kytle, K; Reyes, A et al. (1993) Use of a yeast expression system for the isolation and analysis of drug-resistant mutants of a mammalian phosphodiesterase. Proc Natl Acad Sci U S A 90:11970-4