This program has as its primary objective the design, synthesis and biological evaluation of inhibitors of S-adenosylmethionine (AdoMet)-dependent macromolecular methylations (e.g., nucleic acid and protein). This objective will be accomplished through the design and synthesis of inhibitors of eukaryotic and prokaryotic S-adenosylhomocysteine (AdoHcy) hydrolase. AdoHcy hydrolase is the enzyme that regulates the cellular level of AdoHcy, which is the endogenous inhibitor of AdoMet-dependent methylations. To more rationally design inhibitors of AdoHcy hydrolase, we will study the structure of both the prokaryotic and eukaryotic enzymes, their mechanisms of catalysis, their mechanisms of interaction with inhibitors and the physiological mechanisms by which the activity of these enzymes are regulated in vivo. The biochemical mechanisms by which neplanocin A, a potent inhibitor of AdoHcy hydrolase, produces its antiviral and antibacterial effects will be elucidated. Analogs of neplanocin A will be synthesized and evaluated as inhibitors of eukaryotic (bovine liver) and prokaryotic (A. faecalis) AdoHcy hydrolase and as substrates for eukaryotic (murine L929 cells) and prokaryotic (A. faecalis, B. subtilis) adenosine kinase. The neplanocin analogs will be evaluated for their effects on AdoMet/AdoHcy metabolism in eukaryotes (murine L929, human colon carcinoma cell line HT-29, neuroblastoma N2a cells) and prokaryotes (A. faecalis, B. subtilis) and for their antiviral (vaccinia virus), antitumor (human colon carcinoma cell line HT-29) and antibacterial (A. faecalis, B. subtilis) effects. AdoHcy hydrolase inhibitors (e.g., neplanocin A) will be used to determine the chemical nature of protein carboxylmethylation (e.g., D-aspartyl residues, Beta-linked aspartyl residues) in neuroblastoma cells and to determine the physiological function of this type of post-translational modification in neuronal tissue.
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