The overall goal of this research proposal is to design and synthesize tetrahydrobiopterin (BH4) analogs for the treatment of catecholamine deficiency disorders, such as Parkinson's disease and dystonia. These patients have decreased CSF and brain levels of BH4, an essential cofactor for tyrosine hydroxylase, the rate limiting enzyme in catecholamine biosynthesis. This cofactor, which is synthesized in the tissues in which it is utilized, is also an absolute requirement for tryptophan hydroxylase and phenylalanine hydroxylase, the enzymes which control serotonin biosynthesis and phenylalanine degradation, respectively. BH4 replacement therapy is currently in use for patients with a genetic defect in BH4 biosynthesis, and is in clinical trials for Parkinson's disease and dystonia. However, due to its instability and lipophobicity very high doses are required to achieve a clinical response. These properties are primarily due to the dihydroxypropyl substituent at the 6-position of the tetrahydropterin ring. This group can be replaced with a wide range of substituents without hindering binding to enzyme. A series of analogs will therefore be synthesized which have been modified at the 6-position, with the aim of overcoming the disadvantages of BH4 yet still maintaining good cofactor properties. Both 6R and 6S enantiomers of BH4 can function as cofactors, but there are marked differences in their kinetic and regulatory properties. In particular, the unnatural (6S)-BH4 displays properties which could be highly detrimental in clinical applications. A stereospecific synthesis has therefore been developed which is capable of producing a wide variety of analogs of either 6R or 6S chirality having greater than 99% enantiomeric purity. The goal of this application is to use this procedure to synthesize pure 6R and 6S enantiomers of BH4 analogs and to determine their kinetic and regulatory properties both in vitro and in vivo. The cofactor analogs will be tested for catalytic activity and specificity with tyrosine, tryptophan, and phenylalanine hydroxylases, and their quinoid dihydro-forms as substrates for the cofactor regenerating enzyme, dihydropteridine reductase. The ability to elicit regulatory properties, such as substrate and end-product inhibition, and the capacity to support a constant rate of reaction for an extended time, will also be evaluated. On the basis of in vitro testing, candidates will be selected for in vivo experiments. First, the stimulation of dopamine production by cells in culture will be measured. Secondly, blood levels of the compound will be determined after different routes of administration to rats. Brain levels, and ability to stimulate brain tyrosine hydroxylase will then be assessed after administration by the most effective route.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Bio-Organic and Natural Products Chemistry Study Section (BNP)
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University of South Alabama
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Ayling, J E; Bailey, S W; Boerth, S R et al. (2000) Hyperphenylalaninemia and 7-pterin excretion associated with mutations in 4a-hydroxy-tetrahydrobiopterin dehydratase/DCoH: analysis of enzyme activity in intestinal biopsies. Mol Genet Metab 70:179-88
Rebrin, I; Thony, B; Bailey, S W et al. (1998) Stereospecificity and catalytic function of histidine residues in 4a-hydroxy-tetrahydropterin dehydratase/DCoH. Biochemistry 37:11246-54
Bailey, S W; Ayling, J E (1997) Total chemical synthesis of chirally pure (6S)-tetrahydrofolic acid. Methods Enzymol 281:3-16
Rebrin, I; Bailey, S W; Boerth, S R et al. (1995) Catalytic characterization of 4a-hydroxytetrahydropterin dehydratase. Biochemistry 34:5801-10
Rebrin, I; Bailey, S W; Ayling, J E (1995) Activity of the bifunctional protein 4a-hydroxy-tetrahydropterin dehydratase/DCoH during human fetal development: correlation with dihydropteridine reductase activity and tetrahydrobiopterin levels. Biochem Biophys Res Commun 217:958-65
Bailey, S W; Boerth, S R; Dillard, S B et al. (1993) The mechanism of cofactor regeneration during phenylalanine hydroxylation. Adv Exp Med Biol 338:47-54
Waymire, J C; Ayling, J E; Craviso, G L (1993) Nicotinic cholinergic regulation of tetrahydrobiopterin levels in bovine adrenal chromaffin cells. Adv Exp Med Biol 338:235-8
Bailey, S W; Dillard, S B; Ayling, J E (1991) Role of C6 chirality of tetrahydropterin cofactor in catalysis and regulation of tyrosine and phenylalanine hydroxylases. Biochemistry 30:10226-35