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1 Nucleotide Chemistry and Biochemistry at SLU Michael B. Doughty Associate Professor of Biochemistry Department of Chem & Phys Southeastern Louisiana University 2 Representative Nucleotides 2'-deoxynucleotide 5'-phosphate (DNA form) phosphate ester NH 2 N O -O P O O- nucleotide 5'-phosphate (RNA form) 5’ O N 3’ OH 2'-deoxyribose sugar N N NH 2 base (purine) N O -O P O O- O N OH OH ribose sugar N N 3 DNA Synthesis by Polymerase 3’ DNA template 5’ DNA Pol 3’-OH 5’ DNA primer 3’ DNA template 5’ dNTP PPi 5’ O-N-3’-OH DNA primer (repeat) Inhibitors of DNA polymerase are traditionally used as antimetabolites to treat cancer and DNA virus infections (e.g., Herpes simplex I and II; Cytomegalovirus; Hepatitis B virus, etc.) Novel Template-Competitive DNA Polymerase Inhibitors triphosphate binding region O- 4 NH 2 N O O O P O P O P O OOO- O N N N S template binding region O HO sugar binding region Doughty & Moore, 1996 N3 minor groove subsituent added to stabilize binary Pol-dNTP complex 5 DNA Synthesis by Reverse Transcriptase Reverse transcriptase (RT) is a retroviral enzyme required for incorporation of a viral RNA genome into host DNA RT RNA RNA RT DNA RT DNA DNA DNA Integration and viral particle production RT inhibitors are or could be used to treat retroviral disease (HIV; Hepatitis A and C; Rabies virus; Mumps and Influenza viruses) 6 Template-Competitive RT Inhibitors Required base modifications for RT inhibition N triphosphate binding pocket N H4O9P3O N O R N N S lipophilic template binding pocket O HO minor groove subsituent added to stabilize binary RT-dNTP complex 2'-deoxyribose binding pocket Doughty, Li, & Lin, 2001 7 Structural Comparison of RT and DNA Pol Inhibitors DNA Pol TC inhibitors RT inhibitors Conformationally, the etheno group forces a population where the side chain is bent under the sugar 8 GTP Binding Proteins (G-Proteins) GDP GTP activation GTP GDP + H2O Pi GDP activates cell processes: nerve conduction metabolism secretion replication 9 Conformational Probes of G-Proteins Seifert & Doughty, unpublished 10 Techniques 1. Chemical synthesis of nucleotides. 2. Conformational analysis of nucleotides and enzyme/protein binding sites. 3. Kinetic analysis and other bioassays.