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The Practical Side of Nucleotide
Metabolism
November 29, 2001
The Plan for Today
• Finish up Tuesday’s Leftovers
• Brief Explanation of how dUMP is
converted to dTMP
• Some clinically relevant treatments based
on these pathways that are used to combat:
– Cancer
– Viral Infections
Beyond AMP, GMP and UMP
Purine Biosynthesis
Pyrimidine Biosynthesis
But other forms of these nucleotides are needed
Two Problems
• These are monophosphates (i.e. GMP)- we
need triphosphates (i.e. GTP) for both DNA
and RNA synthesis
• These are ribonucleotides- that’s fine for
RNA but we also need to make DNA
Synthesis of ribonucleotides first
supports the RNA world theory
Specific Kinases Convert NMP
to NDP
Nucleoside
Monophosphates
Nucleoside
Diphosphates
Monophosphate
Kinases
• Monophosphate kinases are specific for the bases
Adenylate Kinase
AMP + ATP
2ADP
Guanylate Kinase
GMP + ATP
GDP + ADP
Conversion of Ribonucleotides
to Deoxyribonucleotides
HOCH
OH
BASE
2
O
5´
H H 1´
4´
H 3´
HO
2´
H
HOCH2 O BASE
OH
5´
H 1´
4´ H
H 3´
RibonucleotideHO
H
H
OH
2´
Reductase
Deoxyribonucleoside
Ribonucleoside
Somehow we need to get rid of this oxygen
Ribonucleotide Reductase
•
•
•
•
•
Catalyzes conversion of NDP to dNDP
Highly regulated enzyme
Regulates the level of cellular dNTPs
Activated prior to DNA synthesis
Controlled by feedback inhibition
dNDP to dNTP (the final step)
• Once dNDPs are generated by
ribonucleotide reductase a general kinase
(nucleoside diphosphate kinase) can
phosphorylate to make the dNTP’s
Nucleoside
diphosphate
kinase
ATP
Beyond dGTP, dATP and dUTP
• So far we’ve made GTP, ATP, and UTP for
incorporation into RNA
• Also dGTP and dATP for incorporation into
DNA
• We still need dCTP for both RNA and DNA
• We also need to generate dTTP for DNA
Synthesis of UTP/CTP
(Easy Problem)
HN
O C
ATP
ATP
Nucleotide Diphosphokinase
HN
O C
O
C
N
H
C
C
N
H
C
C
CH3
H
ATP + Glutamine
CH3
O
H
O
C
NH2
C
H
N
C
C
C
N
H
H
Synthesis of TTP
(Hard Problem)
HN
O C
O
C
N
H
Thymidylate
Synthase
C
C
CH3
H
CH3
HN
O C
O
C
N
H
C
C
CH3
H
• Methyl group is provided by N5,N10-Methylene
tetrahydrofolate
• Dihyrofolate reductase recharges the Dihydrofolate to
N5,N10-Methylene tetrahydrofolate
Role of Folate in dTMP Synthesis
Thymidylate
Synthase
Dihydrofolate
N5,N10-Methylene
tetrahydrofolate
Dihydrofolate
Reductase
Tetrahydrofolate
The Plan for Today
• Finish up Yesterday’s Leftovers
• Brief Explanation of how dUMP is
converted to dTMP
• Some clinically relevant treatments based
on these pathways that are used to combat:
– Cancer
– Viral Infections
Antimetabolites
• Often drugs that inhibit cell growth are used to
combat cancer
• Many of these compounds are analogues of
purine and pyrimidine bases or nucleotides
• Many of these drugs must be activated by cellular
enzymes
• They affect nucleic acid synthesis and tumor
cells tend to be more susceptible since they are
dividing more rapidly
6-Mercaptopurine (6-MP)
• Purine Analogue
• Used clinically to combat
childhood leukemia
• Since 1963 cure rate has
increased from ~4% to
greater than 80%
Inhibitor of Committed Step in
de novo Purine Biosynthesis
PRPP + 6-MP
6-mercaptopurine
ribonucleotide
This reaction is more active in tumor cells
Cytosine Arabinose (araC)
• Metabolized to cytosine arabinose 5’-triphosphate
(araCTP)
• Analogue of CTP
• Incorporated into DNA and inhibits chain synthesis
• Used extensively for acute leukemias
Cytosine NH2
NH2
Cytosine
Ribose N C C H
Arabinose N C C
O C
C
N
H
HOCH2
O
H
H
H
HO
H
OH
O
Differs only in
the sugar
C
N
HOCH2 O
H
OH
H
OH H
C
H
H
Antifolates
• Antifolates interfere
with formation of
dihydrofolate which is
required for:
Thymidylate
Synthase
– dTMP synthesis (today)
– de novo purine
biosynthesis (yesterday) N5,N10-Methylene Dihydrofolate
tetrahydrofolate
X
Dihydrofolate
Reductase
Tetrahydrofolate
Antifolate Agents Mimic Folate
O
Hydroxyurea
H2 N C NHOH
• Specifically inhibits ribonucleotide reductase
• Inhibits DNA synthesis without affecting RNA
synthesis or other nucleotide pools
• Cleared from the body rapidly so not used
extensively in the clinic
Practical Considerations
• Most of these agents are used in combination
therapies
• Many need to be processed in cells to create the
active compound
• Often are not specific for tumor cells but rather for
rapidly dividing tissues
• Multiple modes of drug resistance can and do
develop (Specific or General)
Example of Specific Drug
Resistance: Methotrexate
• Methotrexate works by inhibiting the function of
dihydrolfolate reductase (DHFR)
• Cells develop ways to avoid this block
– Mutations in DHFR that make it bind less tightly to MTX
– Amplication of the DHFR gene (more enzyme activity)
Anti-Viral Therapies
• Target virally infected cells
• Take advantage of aspects of viral metabolism that
differ from normal cellular metabolism
HIV- Human Immunodeficiency Virus
HSV- Herpesvirus
AZT as an Anti-HIV Agent
•
•
•
•
Azido-3’-deoxythymidine
Pyrimidine Analogue
HIV is a retrovirus
RNA genome that is
reverse-transcribed to DNA
• Viral polymerase is
inhibited by AZT
O
C
CH3
HN
C
O C
C
N
H
HOCH2 O
H
H
H
H
N3
H
Acyclovir as an Anti-HSV Agent
•
•
•
•
•
Acyclovar (acycloguanosine)- purine analog
Needs to be phosphorylated to be activated
A viral thymidine kinase catalyzes this reaction
No similar cellular kinase exists
Activated form is a potent DNA polymerase inhibitor
HSV
Polymerase
Unaffected
HSV Polymerase
HSV
Inhibited
kinase
Uninfected
Infected
RIP
The BIG Picture
• GMP, AMP, UMP on…..
• Generation of dTMP
• Common features of clinically relevant
antimetabolites/antifolates
• Antiviral agents- how are they specific for
the virally infected cells?