S-Adenosylhomocysteine (AdoHcy) hydrolase (EC33.1.1) plays an important role in regulating the cellular level of AdoHcy, a potent product inhibitor of S-adenosylmethionine (AdoMet)-dependent transmethylation reactions. Most plant and animal viruses have a 'capped-methylated structure' at the 5'-terminus of their mRNA, which is critical for viral mRNA translation and thus viral replication. Inhibitors of AdoHcy hydrolase elevate cellular levels of AdoHcy, which inhibit the viral mRNA guanine-7 and nucleoside 2'-O-methyltransferases involved in the formation of this 'capped-methylated structure'. By creating a hostile environment in cells (e.g., elevated AdoHcy levels), inhibitors of AdoHcy hydrolase produce a broad-spectrum of antiviral activity. For this reason AdoHcy hydrolase has become an attractive target for the design of antiviral agents. The primary objectives of this research program are twofold: (1) to design and synthesize potent and specific inhibitors of AdoHcy hydrolase and to evaluate their effects on cellular metabolism and viral replication; and (2) to elucidate the role of AdoHcy hydrolase in regulating the cellular homeostasis of AdoHcy and homocysteine and to determine how these alterations in cellular metabolism relate to the inhibition of viral replication. These objectives will be accomplished by: (1) conducting transferred NOE-NMR experiments on recombinant rat liver and human placental AdoHcy hydrolases to determine the optimal structural features for inhibitors and the key amino acids involved in inhibitor binding; (2) generating computer graphics models of the active sites of rat liver and human placental AdoHcy hydrolases; (3) designing and synthesizing mechanism-based inhibitors; (4) determining the molecular mechanism by which these inhibitors inactivate recombinant rat liver and human placental AdoHcy hydrolases; (5) determining their effects on AdoHcy hydrolase activity and on AdoMet/AdoHcy metabolism in cultured cells, and their effects on viral replication; and (6) characterizing, by molecular biological and enzymological techniques, the molecular nature of the different forms of AdoHcy hydrolase and establishing their functional role in regulating homeostasis of AdoHcy and Hcy. Through this multidisciplinary approach to drug design, potent and specific inhibitors of AdoHcy hydrolase, which will potentially be useful in the treatment of viral infections, should be forthcoming.
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