Design of Inhibitors of Epinephrine Biosynthesis

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Epinephrine (Epi) comprises about 5% of central nervous system (CNS) catecholamines and it has been implicated in a number of neuroregulatory processes. We (and others) have shown that inhibitors of phenylethanolamine N-methyltransferase (PNMT, E. C. 2.1.1.28; the terminal enzyme in Epi biosynthesis) can lower blood pressure in spontaneously hypertensive rats. However, all inhibitors examined for blood pressure effects also have high affinity for alpha2-adrenoceptors, which could contribute significantly to the observed pharmacological effects. We now have lead inhibitors that are very selective (very low alpha2 affinity) and have sufficient lipophilicity (using the BBMEC model) to penetrate the blood brain barrier. We have developed microdialysis techniques to allow us to measure the changes in CNS catecholamine levels (DA, NE and Epi) in key brain regions (e.g., hypothalamus) following intraperitoneal administration of PNMT inhibitors We have confirmed that this method can provide results on literature PNMT inhibitors in complete agreement with data obtained from brain homogenate studies. We have cloned and expressed human brain PNMT (hPNMT). The X-ray crystal structure of hPNMT complexed with S-adenosy-L-homocysteine and SK&F 29661 (a competitive inhibitor of PNMT) has recently been determined at 2.4 Angstroms. We propose to take full advantage of these highly integrated results and use structure-based drug design to optimize our lead inhibitors to enhance their PNMT activity and reduce their alpha2 affinity. We have proposed several template skeletons for the structure- based design work based on existing lead inhibitors and the new crystal structure. We also propose to develop a high throughput PNMT screen to search libraries of compounds to identify other leads, particularly the sample libraries of the late Professors E. E. Smissman and M. P. Mertes of the University of Kansas. Lead optimization studies will employ parallel synthesis methods where appropriate. We have developed comparative molecular field analysis (CoMFA) models of both the active site of PNMT and the alpha2 adrenoceptor. The latter model will be refined as new results are obtained as an aid to achieving high selectivity in the new inhibitors. A further aid in the inhibitor design will be site- directed mutagenesis experiments aimed at determining the important amino acid residues for PNMT inhibitor binding. Once we have identified potent inhibitors that are (1) selective (minimal affinity at alpha2 adrenoceptors and other neurotransmitter receptors), (2) lipophilic enough to enter the CNS, and (3) shown to lower CNS Epi levels following i.p. administration, investigation of the effects of these inhibitors on blood pressure and hear rate will be determined. These inhibitors will be the first pharmacological tools available to help determine the function(s) of Epi in the CNS. This project is ripe and ready to burst forth with important new results.