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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029332-28
Application #
6329626
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Schwab, John M
Project Start
1980-12-01
Project End
2002-08-31
Budget Start
2000-12-01
Budget End
2001-11-30
Support Year
28
Fiscal Year
2001
Total Cost
$296,751
Indirect Cost
Name
University of Kansas Lawrence
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
072933393
City
Lawrence
State
KS
Country
United States
Zip Code
66045
Cai, Sumin; Fang, Jianwen; Li, Qing-Shan et al. (2010) Comparative kinetics of cofactor association and dissociation for the human and trypanosomal S-adenosylhomocysteine hydrolases. 3. Role of lysyl and tyrosyl residues of the C-terminal extension. Biochemistry 49:8434-41
Li, Qing-Shan; Cai, Sumin; Fang, Jianwen et al. (2009) Evaluation of NAD(H) analogues as selective inhibitors for Trypanosoma cruzi S-adenosylhomocysteine hydrolase. Nucleosides Nucleotides Nucleic Acids 28:473-84
Cai, Sumin; Li, Qing-Shan; Fang, Jianwen et al. (2009) The rationale for targeting the NAD/NADH cofactor binding site of parasitic S-adenosyl-L-homocysteine hydrolase for the design of anti-parasitic drugs. Nucleosides Nucleotides Nucleic Acids 28:485-503
Li, Qing-Shan; Cai, Sumin; Fang, Jianwen et al. (2008) Comparative kinetics of cofactor association and dissociation for the human and trypanosomal S-adenosylhomocysteine hydrolases. 2. The role of helix 18 stability. Biochemistry 47:4983-91
Wnuk, Stanislaw F; Sacasa, Pablo R; Lewandowska, Elzbieta et al. (2008) Synthesis of 5'-functionalized nucleosides: S-Adenosylhomocysteine analogues with the carbon-5'and sulfur atoms replaced by a vinyl or halovinyl unit. Bioorg Med Chem 16:5424-33
Hu, Chen; Fang, Jianwen; Borchardt, Ronald T et al. (2008) Molecular dynamics simulations of domain motions of substrate-free S-adenosyl- L-homocysteine hydrolase in solution. Proteins 71:131-43
Cai, Sumin; Li, Qing-Shan; Borchardt, Ronald T et al. (2007) The antiviral drug ribavirin is a selective inhibitor of S-adenosyl-L-homocysteine hydrolase from Trypanosoma cruzi. Bioorg Med Chem 15:7281-7
Li, Qing-Shan; Cai, Sumin; Borchardt, Ronald T et al. (2007) Comparative kinetics of cofactor association and dissociation for the human and trypanosomal S-adenosylhomocysteine hydrolases. 1. Basic features of the association and dissociation processes. Biochemistry 46:5798-809
Nowak, Ireneusz; Robins, Morris J (2007) Synthesis of 3'-deoxynucleosides with 2-oxabicyclo[3.1.0]hexane sugar moieties: addition of difluorocarbene to a 3',4'-unsaturated uridine derivative and 1,2-dihydrofurans derived from D- and L-xylose1. J Org Chem 72:3319-25
Nowak, Ireneusz; Robins, Morris J (2007) Trifluoromethylation of alkenyl bromides and iodides (including 5-iodouracils) with (CF3)2Hg and Cu (""trifluoromethylcopper""). J Org Chem 72:2678-81

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