The overall goals of the proposed research project are to address fundamental questions related to the enzymology of L-pipecolic acid metabolism in the mammalian brain and to develop potent, specific inhibitors of its degradation. Such inhibitors represent a potential new means for developing anticonvulsive agents and for studying GABA receptor complexes. L-Pipecolic (L-PA) acid is a six-carbon cyclic amino acid, the higher homologue of L-proline, and is a minor product of lysine metabolism in various organisms and most mammalian tissues. The notable exception is the brain, where lysine is primarily degraded to L-PA which has been shown to possess both neuromodulating and anticonvulsive properties. A large body of evidence supports an interaction of L-pipecolate and GABAergic transmission, either at GABA receptors or through binding to its own receptor and exerting an allosteric effect on the GABA complex. Hence, a primary objective of this proposal is to define the features of the key enzyme involved in L-pipecolic acid catabolism in the brain and use this information to develop specific inactivators of it, thereby decreasing L- PA degradation and elevating its concentration and neurological effects. In liver and kidney, the first step of L-PA degradation is known to be species dependent, and is catalyzed by either a flavin-containing peroxisomal oxidase or mitochondrial dehydrogenase. Rhesus monkey liver L- pipecolate oxidase has been purified, but nothing is known about the enzymology of L-PA metabolism in the brain or about the traits of the mitochondrial enzyme from any source. Thus, the first specific goals of this project will be to isolate and fully characterize mitochondrial L- pipecolate oxidizing enzymes from rabbit kidney and brain, and the peroxisomal enzyme from primate brain. This will generate data leading to a better understanding of L-PA metabolism in general and presents a unique opportunity to study different enzymes catalyzing the same reaction in different mammalian species. Complete analysis of these enzymes will include: determining physical and kinetic properties; detailed cofactor studies; and stereochemical and mechanistic relatedness of the enzymes isolated from the different species and organelles. The second specific aim of this project involves designing and constructing modified substrates and potent inhibitors of L-PA oxidation. The focus will be on mechanism-based inactivators designed to exploit features characteristic of flavin-dependent amine oxidases. These inactivators will serve as valuable probes of the mechanisms and active site environments of the various L-pipecolate oxidizing enzymes. Inhibitors designed to develop highly reactive radical or electrophilic species could form covalent adducts with the target enzymes and permit active site sequencing and comparisons. Potent inactivators may unveil a new route to designing therapeutically useful anticonvulsants and the pipecolate analogs developed in this work may serve as valuable pharmacological ligands for studying and classifying GABA receptors.
| Zabriskie, T M; Jackson, M D (2000) Lysine biosynthesis and metabolism in fungi. Nat Prod Rep 17:85-97 |
| Ho, B; Zabriskie, T M (1998) Epoxide derivatives of pipecolic acid and proline are inhibitors of pipecolate oxidase. Bioorg Med Chem Lett 8:739-44 |
| Edwards, J P; West, S J; Pooley, C L et al. (1998) New nonsteroidal androgen receptor modulators based on 4-(trifluoromethyl)-2(1H)-pyrrolidino[3,2-g] quinolinone. Bioorg Med Chem Lett 8:745-50 |
| Zabriskie, T M (1996) Mechanism-based inhibition of L-pipecolate oxidase by 4,5-dehydro-L-pipecolic acid. J Med Chem 39:3046-8 |
