the practical side of nucleotide metabolism november 29, 2001
TRANSCRIPT
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
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?