Transition State Analysis in Design of Potential Phosphodiesterase Inhibitors

Edward Ollie, Masaru Miyagi, Daniel Kellerman, Michael Harris

University of Florida (E. Ollie, M. Harris.)
Case Western Reserve University (M. Miyagi, D. Kellerman)

Cyclic nucleotide phosphodiesterase (PDE) enzymes degrade the cyclic nucleotides 3’,5’-cAMP and 3,5"-cGMP.  A diverse array of processes rely on a cycle of synthesis and degradation of these second messengers; thus inhibition of PDE enzymes is an attractive goal for drug discovery. Enzymes catalyze reactions by stabilizing the transition state, thus binding transition states more tightly than reactant or product. Studying the transition states involved in these reactions could allow for design of novel PDE inhibitors that mimic transition state structure or improvement on existing models. Kinetic isotope effect (KIE) experiments, which measure changes in rate constants due to substitution of heavy isotopes at a particular position, are a valuable tool for understanding catalytic mechanism and determining transition state bonding. The aim of this project is the development of a method to measure solvent H218O nucleophile isotope effects (18knuc) on the enzymatic hydrolysis of 3’,5’-cAMP to 5’AMP by PDE4D2, a cAMP-specific PDE, via tandem time of flight mass spectrometry. Analysis of the solvent 18knuc provides important insights into transition state bonding interactions and is essential for the larger goal of more accurately modelling transition state structure and design or improvement of PDE inhibitors.