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.; 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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Bio-Organic and Natural Products Chemistry Study Section (BNP)
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Barouch, Winifred
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University of Kansas Lawrence
Schools of Pharmacy
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Wu, Qian; McLeish, Michael J (2013) Kinetic and pH studies on human phenylethanolamine N-methyltransferase. Arch Biochem Biophys 539:1-8
Drinkwater, Nyssa; Vu, Hoan; Lovell, Kimberly M et al. (2010) Fragment-based screening by X-ray crystallography, MS and isothermal titration calorimetry to identify PNMT (phenylethanolamine N-methyltransferase) inhibitors. Biochem J 431:51-61
Drinkwater, Nyssa; Gee, Christine L; Puri, Munish et al. (2009) Molecular recognition of physiological substrate noradrenaline by the adrenaline-synthesizing enzyme PNMT and factors influencing its methyltransferase activity. Biochem J 422:463-71
Wu, Qian; Caine, Joanne M; Thomson, Stuart A et al. (2009) Time-dependent inactivation of human phenylethanolamine N-methyltransferase by 7-isothiocyanatotetrahydroisoquinoline. Bioorg Med Chem Lett 19:1071-4
Georgieva, Polina; Wu, Qian; McLeish, Michael J et al. (2009) The reaction mechanism of phenylethanolamine N-methyltransferase: a density functional theory study. Biochim Biophys Acta 1794:1831-7
Grunewald, Gary L; Seim, Mitchell R; Bhat, Seema R et al. (2008) Synthesis of 4,5,6,7-tetrahydrothieno[3,2-c]pyridines and comparison with their isosteric 1,2,3,4-tetrahydroisoquinolines as inhibitors of phenylethanolamine N-methyltransferase. Bioorg Med Chem 16:542-59
Gee, Christine L; Drinkwater, Nyssa; Tyndall, Joel D A et al. (2007) Enzyme adaptation to inhibitor binding: a cryptic binding site in phenylethanolamine N-methyltransferase. J Med Chem 50:4845-53
Grunewald, Gary L; Seim, Mitchell R; Regier, Rachel C et al. (2007) Exploring the active site of phenylethanolamine N-methyltransferase with 1,2,3,4-tetrahydrobenz[h]isoquinoline inhibitors. Bioorg Med Chem 15:1298-310
Grunewald, Gary L; Seim, Mitchell R; Lu, Jian et al. (2006) Application of the Goldilocks effect to the design of potent and selective inhibitors of phenylethanolamine N-methyltransferase: balancing pKa and steric effects in the optimization of 3-methyl-1,2,3,4-tetrahydroisoquinoline inhibitors by beta-fluorinatio J Med Chem 49:2939-52
Grunewald, Gary L; Seim, Mitchell R; Regier, Rachel C et al. (2006) Comparison of the binding of 3-fluoromethyl-7-sulfonyl-1,2,3,4-tetrahydroisoquinolines with their isosteric sulfonamides to the active site of phenylethanolamine N-methyltransferase. J Med Chem 49:5424-33

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