S-Adenosyl-L-homocysteine (AdoHcy) hydrolase (EC 3.3.1.1.) in mammalian cells and parasites plays a key role in regulating the intracellular levels of AdoHcy and homocysteine (Hcy) by catalyzing their interconversion (AdoHcy reversible reaction Ado plus Hcy). This NAD+-dependent enzyme exhibits unique structural and catalytic features which are worthy of further investigation. The human and parasite enzymes are also attractive therapeutic targets because: (i) clinical data have shown that elevated plasma levels of Hcy (Hcymia) are a risk factor in coronary artery disease; therefore, human AdoHcy hydrolase is an attractive target for the design of drugs to reduce plasma Hcy levels thus reducing a patient's risks of developing cardiovascular disease; and (ii) the intracellular levels of AdoHcy regulate AdoMet-dependent methyltransferases that are crucial for replication of certain viruses (Ebola, rabies, respiratory syncytical) and parasites [Leishmania (L.) donovani, Trypanosoma (T.) cruzi]; therefore, the human AdoHcy hydrolase is an attractive target for the design of antiviral agents and the parasite enzymes are attractive targets for the design of antiparasitic agents. During the next grant period, our primary objectives will include: (i) elucidating the relationships between the structure and catalytic mechanism of the human enzyme by conducting X-ray crystallographic, site-directed mutagenesis, fluorescence spectroscopy, and molecular modeling studies as well as designing and synthesizing new structural and mechanistic probes; and (ii) identifying through high through-put screening and structure-based drug design specific inhibitors of parasite AdoHcy hydrolases as potential antiparasitic agents. During the last grant period, our laboratories have made the following significant advances that will facilitate completion of those objectives: (i) determined the X-ray crystal structures of several inhibitor-inactivated forms of the recombinant human enzyme; (ii) developed the experimental protocols to use site- directed mutagenesis, fluorescence spectroscopy, and molecular modeling to probe the structure and catalytic mechanism of this enzyme; (iii) designed and synthesized type II mechanism-based inhibitors (covalent inactivation) of the human enzyme which have proven useful as structural and mechanistic probes; (iv) developed a high through-put screen which can now be used to evaluate potential inhibitors of the parasitic enzymes; and (v) developed a plasmid for overexpression of the L. donovani enzyme which will provide large quantities of this enzyme for studies that will serve as the basis for structure-based design of antiparasitic agents.
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