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 2-hexadecenal and ethanolamine phosphate. 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 the human or mouse gene. 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 causes rapid translocation of calcium from intracellular stores. Blocking 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 that sphingosine-1-phosphate lyase is a pyridoxal phosphate-dependent enzyme. We are synthesizing analogs of sphingosine that have these groups in the 2-position. 2-Vinyl diphydrazine and 2-hydrazino-dihydrosphingosine has already been prepared and characterized. The 2-vinyl analog is efficiently phosphorylated by a rat liver cytosolic preparation that contains sphingosine kinase. Experiments are in progress to determine the extent of inhibition of sphingosine-1-phosphate lyase caused by treatment of cultured mammalian cells with 2-vinyl-dihydrosphingosine. Synthesis of 2-vinyl and 2-difluoromethyl analogs of the product ethanolamine phosphate are in progress. In vitro assays have been developed in our section for both the kinase and lyase reactions.
