Phenylethanolamine N-methyltransferase (PNMT, E. C. 2.1.1.28) is the enzyme that catalyzes the terminal step in the biosynthesis of epinephrine (Epi). Epi comprises about 5 percent of central nervous system (CNS) catecholamine content and it has been implicated in a number of neuroregulator processes in the brain. It was demonstrated (our laboratory and others) that inhibitors of PNMT can lower blood pressure in spontaneously hypertensive rats However, all of the inhibitors presently available have high affinity for a2-adrenergic receptors, which could contribute significantly to the observed pharmacological effects. A number of probes to determine the active site binding requirements for PNMT and for the a2-adrenoceptor have been synthesize in our laboratory and used to develop computer graphics models (comparative molecular field analysis; CoMFA) of the two sites. These models have 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 a2-adrenoceptor. The results from the evaluation of these ligands will be used to refine our computer model and improve its ability to aid in the design of a potent and selective inhibitor of PNMT with sufficient lipophilicity to cross the blood brain barrier. Human brain PNMT (hPNMT) has recently been cloned and expressed (in collaboration with M. McLeish) and thre different crystals of hPNMT in the presence of three PNMT inhibitors with different binding characteristics have been grown (in collaboration with J. Martin); the crystal structures are awaiting solution. A combination of computer modeling, protein crystallography (structure-based ligand design), site-directed mutagenesis and homology modeling of the active site of hPNMT will allow us to develop more selective and potent inhibitors. A high throughput screen for PNMT (based on coupling with AdoHcy hydrolase, in collaboration with R. Borchardt) will be developed and used to screen unique libraries of structurally diverse compounds in the Smissman and Mertes sample collections at the U. of Kansas. Leads will be optimized with parallel synthesis techniques where appropriate. Microdialysis experiments with capillary electrophoresis (in collaboration with S. Lunte) will allow measurement of the effects on CNS Epi levels of new PNMT inhibitors in vivo in conscious rats. Results from all sub-projects will act synergistically to lead to the synthesis of a highly potent and selective inhibitor of PNMT, which would be useful as a pharmacological tool to probe the role(s) played by Epi i the CNS, and, in particular, potentially identify a new mechanism for drug treatment of hypertension.
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