Transcript Nucleotide Chemistry and Biochemistry at SLU

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Nucleotide Chemistry and
Biochemistry at SLU
Michael B. Doughty
Associate Professor of Biochemistry
Department of Chem & Phys
Southeastern Louisiana University
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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
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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
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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)
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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
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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
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GTP Binding Proteins (G-Proteins)
GDP


GTP

activation
GTP

GDP
+

H2O

Pi
GDP

activates cell processes:
nerve conduction
metabolism
secretion
replication
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Conformational Probes of G-Proteins
Seifert &
Doughty,
unpublished
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Techniques
1. Chemical synthesis of nucleotides.
2. Conformational analysis of nucleotides and
enzyme/protein binding sites.
3. Kinetic analysis and other bioassays.