This project covers the synthesis of enzyme substrates and enzyme inhibitors which are used to study sphingolipid metabolism. The major ongoing project is the design and synthesis of inhibitors of sphingosine- 1-phosphate lyase. This enzyme catalyzes the last step in the degradation of sphingosine: the cleavage of sphingosine phosphate to palmitaldehyde and ethanolamine phosphate. Very little is known about this enzyme and about the role(s) of free sphingosine in vivo. Inhibitors of sphingosine-1 phosphate lyase could advance understanding sphingosine catabolism in two ways. The preparation of radiolabeled irreversible inhibitors would aid in the isolation and purification of the enzyme. This could lead to partial sequence determination, and ultimately to cloning of the human or mouse gene. No sequence data are currently available on sphingosine-1-phosphate from any organism. A second use for enzyme inhibitors would be to provide information on the biological effects of blocking sphingosine catabolism in vivo. Sphingosine has been reported to be an inhibitor of protein kinase C, and sphingosine-1-phosphate lyase would lead to accumulation of these two compounds, possibly causing profound changes in cellular regulation. Our approach to the design of inhibitors is based upon the fact the sphingosine-1-phosphate lyase is a pyridoxal phosphate-dependent enzyme. Three initial synthetic targets were chosen for synthesis. They are the 2-difluoromethyl and the 2-vinyl derivatives of dihydrosphingosine, and 1,3-dihydroxy-2-hydrazino-octadecane. Progress has been made on all tree syntheses, and small samples of the hydrazine analog are now available for characterization and testing. Initial assays will use Tetrahymena furgasoni as the enzyme source. The degradation of sphingosine in Tetrahymena occurs by the same two-step pathway (via sphingosine kinase and sphingosin-1-phosphate lyase) that is utilized by mammals. We are now culturing this organism in our laboratory, and are developing a bioassay for sphingosine catabolism that can be used to screen inhibitors. High specific activity [3H]-dihydrosphingosine that was previously synthesized in this laboratory is being used in these bioassays. Initial studies will follow sphingosine catabolism in whole cells of this protozoan. Subsequently studies will use sphingosine-1- phosphate lyase that will be isolated from Tetrahymena and mammalian sources.
