The enzymatic transfer of a methyl group from S-adenosylmethionine (AdoMet) to an acceptor molecule, i.e., biological transmethylation, is an ubiquitous set of reactions involved in regulating a diverse array of physiologically important processes. The activities of all AdoMet- dependent methyltransferases are regulated by the intracellular level of S-adenosyl-L-homocysteine (AdoHcy), a product inhibitor of AdoMet- dependent transmethylations. In all eukaryotic cells, the intracellular level of AdoHcy is regulated by the enzyme AdoHcy hydrolase (EC 3.3.1.1), which catalyzes the degradation of AdoHcy to adenosine (Ado) and homocysteine (Hcy) through a mechanism involving an NAD+-dependent oxidation (oxidative activity) of the 3'-hydroxyl group of AdoHcy followed by elimination of Hcy to form 4',5'-dehydro-3-keto-Ado. Addition of water at the 5'-position (hydrolytic activity) of this tightly bound intermediate, followed by NADH-dependent reduction results in the formation of Ado. In recent years, a number of potent and specific mechanism-based inhibitors of AdoHcy hydroxylase have been designed and synthesized. These inhibitors function by serving as substrates for the 'oxidative activity' of this enzyme converting the enzyme from its active form (NAD+) to its inactive form (NADH) (Type I mechanism-based inhibitors). In addition to being useful chemical tools to study the biological functions of AdoHcy hydrolase and AdoMet- dependent methylations, these AdoHcy hydrolase inhibitors have shown pharmacological effects of clinical significance including antiviral (cytomegalovirus, respiratory syncytical virus, rabies) and antiparasitic (Leishmania, Plasmodium, Trypansoma) effects. In spite of the crucial role this enzyme plays in regulating biological processes and its attractiveness as a target for drug design, little is known about the terteriary and quaternary structures of this protein. In addition, while many potent and specific inhibitors of AdoHcy hydrolase have been identified in recent years, they all function by the same Type I mechanism. Therefore, the major objectives of this research program are twofold: (a) to determine the structural features and the inhibitor specificities of recombinant human placental and Leishmania (L) donovani AdoHcy hydrolases; and (b) to use this information to design and synthesize mechanism-based inhibitors which exploit the 'hydrolytic activity' of the enzyme and are specific for the human enzyme (potential antiviral agents) or the parasite enzyme (potential antiparasitic agents). These objectives will be accomplished by: (a) determining the structural features of recombinant human placental and L. donovani AdoHcy hydrolases using chemical, molecular biological, spectroscopic, biophysical and computer modeling techniques; (b) designing, synthesizing and biochemically evaluating new inhibitors of AdoHcy hydrolase which utilize the enzyme's 'hydrolytic activity'; (c) elucidating the mechanisms by which these inhibitors inactivate the human placental and L. donovani AdoHcy hydrolases; and (d) determining the antiviral and antiparasitic effects of these inhibitors. Through this multidisciplinary approach to drug design, potent and specific inhibitors of the human and L. donovani AdoHcy hydrolases, which may have clinical potential, should be forthcoming.
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