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

NucleosideMonophosphates

NucleosideDiphosphates

MonophosphateKinases

• Monophosphate kinases are specific for the bases

AMP + ATP 2ADP

GMP + ATP GDP + ADP

Adenylate Kinase

Guanylate Kinase

Conversion of Ribonucleotides to Deoxyribonucleotides

OH

HHO

H

H

HOCH2

OH

OH

1´

2´3´

4´5´O

H

HHO

H

H

HOCH2 OH

H

1´

2´3´

4´

5´BASE BASE

Deoxyribonucleoside Ribonucleoside

Somehow we need to get rid of this oxygen

RibonucleotideReductase

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

ATP

Nucleoside diphosphate

kinase

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)

Nucleotide Diphosphokinase

ATP ATP

NC

CC

N

CH

O

H

H

NH2

NC

CC

HN

C

O

CH3

HO

H

NC

CC

HN

C

O

CH3

HO

H

ATP + Glutamine

Synthesis of TTP(Hard Problem)

NC

CC

HN

C

O

CH3

HO

H

NC

CC

HN

C

O

CH3

HO

H

ThymidylateSynthase

• Methyl group is provided by N5,N10-Methylene tetrahydrofolate

• Dihyrofolate reductase recharges the Dihydrofolate to N5,N10-Methylene tetrahydrofolate

CH3

Role of Folate in dTMP Synthesis

DihydrofolateReductase

ThymidylateSynthase

DihydrofolateN5,N10-Methylene tetrahydrofolate

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%

PRPP + 6-MP6-mercaptopurine

ribonucleotide

Inhibitor of Committed Step inde novo Purine Biosynthesis

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 Arabinose

Differs only in the sugar

NC

CC

N

CH

O

H

NH2

HOH

H

H

HO

OH

HOCH2N

C

CC

N

CH

O

H

NH2

HOCH2

OH

OHH

H

O

H

Cytosine Ribose

Antifolates

• Antifolates interfere with formation of dihydrofolate which is required for:– dTMP synthesis (today)

– de novo purine biosynthesis (yesterday)

ThymidylateSynthase

DihydrofolateN5,N10-Methylene tetrahydrofolate

Tetrahydrofolate

DihydrofolateReductaseX

Antifolate Agents Mimic Folate

Hydroxyurea

• 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

H2N C

O

NHOH

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)

• Target virally infected cells• Take advantage of aspects of viral metabolism that

differ from normal cellular metabolism

Anti-Viral Therapies

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

NC

CC

HN

C

O

HO

H

H

H

HO

HN3

CH3

HOCH2

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

Uninfected Infected

HSV

HSV

HSV

kinase

PolymeraseUnaffected

PolymeraseInhibited

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?