The Practical Side of Nucleotide Metabolism

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The Practical Side of Nucleotide Metabolism

• November 29, 2001

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The Plan for Today

• Finish up Tuesdays 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

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Beyond AMP, GMP and UMP
Purine Biosynthesis
Pyrimidine Biosynthesis
GMP
AMP
UMP
But other forms of these nucleotides are needed
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Two Problems

• These are monophosphates (i.e. GMP)- we need
  triphosphates (i.e. GTP) for both DNA and RNA
  synthesis
• These are ribonucleotides- thats fine for RNA
  but we also need to make DNA

Synthesis of ribonucleotides first supports the
RNA world theory
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Specific Kinases Convert NMP to NDP
Nucleoside Monophosphates
Nucleoside Diphosphates
Monophosphate Kinases
NMP
NDP

• Monophosphate kinases are specific for the bases

Adenylate Kinase
Guanylate Kinase
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Conversion of Ribonucleotides to
Deoxyribonucleotides
BASE
BASE
Ribonucleotide Reductase
Deoxyribonucleoside
Ribonucleoside
Somehow we need to get rid of this oxygen
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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

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dNDP to dNTP (the final step)

• Once dNDPs are generated by ribonucleotide
  reductase a general kinase (nucleoside
  diphosphate kinase) can phosphorylate to make the
  dNTPs

Nucleoside diphosphate kinase
CDP
CTP
ATP
dGDP
dGTP
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Beyond dGTP, dATP and dUTP

• So far weve 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

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Synthesis of UTP/CTP (Easy Problem)
UMP
UDP
UTP
ATP
ATP
Nucleotide Diphosphokinase
ATP Glutamine
CTP
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Synthesis of TTP(Hard Problem)
Thymidylate Synthase
dUMP
dTMP
CH3

• Methyl group is provided by N5,N10-Methylene
  tetrahydrofolate
• Dihyrofolate reductase recharges the
  Dihydrofolate to N5,N10-Methylene tetrahydrofolate

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Role of Folate in dTMP Synthesis
Dihydrofolate Reductase
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The Plan for Today

• Finish up Yesterdays 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

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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

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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
6-mercaptopurine ribonucleotide
PRPP 6-MP
This reaction is more active in tumor cells
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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 Ribose
Cytosine Arabinose
Differs only in the sugar
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Antifolates

• Antifolates interfere with formation of
  dihydrofolate which is required for
• dTMP synthesis (today)
• de novo purine biosynthesis (yesterday)

Thymidylate Synthase
dUMP
dTMP
Dihydrofolate
N5,N10-Methylene tetrahydrofolate
Tetrahydrofolate
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Antifolate Agents Mimic Folate
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Hydroxyurea

• Specifically inhibits ribonucleotide reductase

NDP
dNDP

• Inhibits DNA synthesis without affecting RNA
  synthesis or other nucleotide pools
• Cleared from the body rapidly so not used
  extensively in the clinic

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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)

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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)

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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
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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

DNA
RNA
Protein
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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

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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?

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