Pyrimidine Metabolism for Cancer Therapy

1. ATP? Depends on the system….

4. 1 ATP – Glucose to G-6-P   Ribose-5-P 1 ATP – But 2 high energy bonds to get to PRPP 4 ATP – In vertebrates to get onto IMP from PRPP 5 ATP – In prokaryotes to get onto IMP from PRPP The pentose phosphate shunt can go back through glycolytic pathways, utilizing another ATP (Fructose-6-P to Fructose-1,6,bisP and then onto Ribose-5-P), but not necessary to generate Ribose-5-P)

5. Pyrimidine Metabolism and Cancer Therapy

6. LEARNING OBJECTIVES • Nomenclature of pyrimidines* • Key features of biosynthetic pathway and origin of atoms in pyrimidine ring • Basis of chemotherapy using nucleotide analogs *Key words are highlighted in yellow

7. Pyrimidine Synthesis • The pyrimidine ring is completely synthesized, then attached to a ribose-5-phosphate donated by PRPP • Source of carbons and nitrogens less diverse than with purines.

8. (Carbamoyl-P)

10. Urea Synthesis Pyrimidine Synthesis

11. Carbamoyl Phosphate Synthase and Channeling of Intermediates Miles et al. J. Biol. Chem. 274, 12193-12199 (1999)

12. Enzymatic functions from one large protein (215,000 Mr) Enzymatic functions from one large protein

13. What to Know Compare and contrast CPS I and CPS II CPSII, aspartate transcarbamoylase, and dihydroorotase are three enzymatic functions in one protein. Oratatephosphoribosyltransferase and OMP decarboxylase are two enzymatic functions in one protein. Deficiency leads to Orotic Aciduria. Know symptoms and how to treat. Orotate is made and then attached to a PRPP.

14. Nitrogen Donor (again!) UMP UDP UTP Uridine monophosphate kinase Uridine diphosphate kinase Uridine Cytidine

15. Thymidylate synthase is a major target for anti-cancer therapy. 5-fluorouracil Methotrexate also inhibits this reaction See Figs 26.25-26.27 in reading for more details

16. Regulation of Pyrimidine Synthesis

17. Pyrimidine Breakdown

18. AMP ADP ATP Adenosine monophosphate kinase Adenosine diphosphate kinase GMP GDP GTP Guanosine monophosphate kinase Guanosine diphosphate kinase UDP UTP Uridine monophosphate kinase Uridine diphosphate kinase dUMP dUDP CTP Thymidylate synthase Thymidine diphosphate kinase dTMP dTDP dTTP Thymidine monophosphate kinase De novo purine synthesis IMP De novo pyrimidine synthesis UMP

19. Very Important! Ribonucleotides to Deoxyribonucleotides

20. Ribonucleotides to Deoxyribonucleotides

21. Logic For Deoxynucleotide Synthesis (Fig 26.24) in reading • High [ATP], plenty of energy, ok to make DNA • High [ATP] means the ribonucleotide reductase is active (ON) • ATP in specificity site S favors CDP or UDP in catalytic site C  [dCDP] and [dUDP] ↑ • dCDP and dUDP become metabolized to dTTP (thus justifying the synthesis of dUMP even though it does not get incorporated into DNA) • As [dTTP]↑, it will begin to occupy specificity site favoring GDP in catalytic site, thus leading to [dGP]↑  [dGTP]↑ • As [dGTP]↑ it begins to occupy specificity site and thus favor ADP in catalytic site, leading to [dADP]↑  which will replace ATP in activity site and turn enzyme off

22. Uracil Analogues in Cancer Therapy Uracil 5-fluorouracil β-D-arabinofuranosylcytosine (Ara-C)

23. Thymidylate Synthase dUMP dTMP O F HN O N 2-O3POH2C dFUMP O HO • Cancer Therapy with 5-fluorouracil • 5-FU is typically given with thymidine to boost its effectiveness • 5-FU is converted to dFUMP • dFUMP inhibits Thymidylate synthase • In cancer cells, 5-FU is also incorporated into RNA • 5-FU in RNA is detrimental to cancer cells • Inhibition of Thymidylate synthase is detrimental to both cancer and normal cells • So, administration of thymidine protects both normal and cancer cells, but 5-FU in cancer cells kill them

24. Thymine Analogues in Cancer Therapy Thymidine Bromodeoxyuridine Trifluorothymidine

25. Purine Analogues in Cancer Therapy Hypoxanthine 6-mercaptopurine 6-thioguanine Purines are used as free bases or nucleosides, as nucleotides are poorly transported across the membrane.