DESIGN OF INHIBITORS OF EPINEPHRINE BIOSYNTHESIS

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Phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28) is the enzyme that catalyzes the terminal step in the biosynthesis of epinephrine. Increased levels of this enzyme are found during periods of stress and in hypertension. Epinephrine has been implicated in a number of neuroregulatory processes in the brain. We (and others) have demonstrated that inhibitors of PNMT can lower blood pressure in spontaneously hypertensive rats. However, all of the inhibitors presently available have high affinity for alpha2-adrenergic receptors that could contribute significantly to the observed pharmacological effects. We have previously synthesized a number of probes to determine the active site binding requirements for PNMT and have used this information to develop a computer graphics model of the active site. The model has been used to design new ligands for synthesis that, based on preliminary studies, have the potential of exhibiting the desired level of selectivity for the PNMT active site over the alpha-adrenergic receptor. These new ligands will be synthesized and evaluated for their affinity and selectivity for PNMT. The results will be incorporated into an improved computer model of the active site of PNMT, and will also be used to construct a complementary model of the alpha-adrenergic receptor. In addition, we will continue our highly successful studies employing the transferred nuclear Overhauser enhancement method to determine the conformation of a few key inhibitors and substrates when bound to the active site of PNMT. The nmr conformational information will allow improvement of the predictive utility of the computer model. We will also do affinity labeling of amino acid residues at the active site and use this information, in conjunction with knowledge-based homology modeling, to construct a possible three- dimensional model of the enzyme. A sufficient quantity of pure PNMT will be obtained from bovine adrenals to allow attempts at crystallization of the enzyme and a determination of its X-ray crystal structure. Molecular biology experiments are proposed that will allow us to obtain human adrenal PNMT and possibly human brain PNMT in quantities sufficient to evaluate potential inhibitors using the more relevant human enzyme and also to attempt crystallization and X-ray crystal structure determination of it. Results from all sub-projects will be combined in a synergistic fashion to lead to the synthesis of a highly potent and selective inhibitor of PNMT. Such an inhibitor would be a useful pharmacological tool to probe the role played by epinephrine in the central nervous system, and, in particular, potentially identify a new mechanism for drug treatment of hypertension.