1. The Practical Side of Nucleotide Metabolism November 29, 2001

2. 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

3. Beyond AMP, GMP and UMP Purine BiosynthesisPyrimidine Biosynthesis But other forms of these nucleotides are needed

4. 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

5. Specific Kinases Convert NMP to NDP Nucleoside Monophosphates Nucleoside Diphosphates Monophosphate Kinases Monophosphate kinases are specific for the bases AMP + ATP2ADP GMP + ATPGDP + ADP Adenylate Kinase Guanylate Kinase

6. Conversion of Ribonucleotides to Deoxyribonucleotides 1´ 2´3´ 4´ 5´ 1´ 2´3´ 4´ 5´ BASE DeoxyribonucleosideRibonucleoside Somehow we need to get rid of this oxygen Ribonucleotide Reductase

7. 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

8. 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

9. 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

10. Synthesis of UTP/CTP (Easy Problem) Nucleotide Diphosphokinase ATP ATP + Glutamine

11. Synthesis of TTP (Hard Problem) Thymidylate Synthase Methyl group is provided by N 5,N 10 -Methylene tetrahydrofolate Dihyrofolate reductase recharges the Dihydrofolate to N 5,N 10 -Methylene tetrahydrofolate CH 3

12. Role of Folate in dTMP Synthesis Dihydrofolate Reductase Thymidylate Synthase Dihydrofolate N 5,N 10 -Methylene tetrahydrofolate Tetrahydrofolate

13. 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

14. 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

15. 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-MP 6-mercaptopurine ribonucleotide Inhibitor of Committed Step in de novo Purine Biosynthesis This reaction is more active in tumor cells

16. 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 Cytosine Ribose

17. Antifolates Antifolates interfere with formation of dihydrofolate which is required for: –dTMP synthesis (today) –de novo purine biosynthesis (yesterday) Thymidylate Synthase Dihydrofolate N 5,N 10 -Methylene tetrahydrofolate Tetrahydrofolate Dihydrofolate Reductase X

18. Antifolate Agents Mimic Folate

19. 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

20. 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)

21. 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)

22. 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

23. 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

24. 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 UninfectedInfected HSV kinase Polymerase Unaffected Polymerase Inhibited RIP

25. 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?