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Improved Fluoropyrimidine Chemotherapy

Improved Fluoropyrimidine Chemotherapy. William H. Gmeiner, Ph.D. Professor and Chairman Department of Biochemistry, WFUSM. Colon Cancer: 2 nd Leading Cause of Cancer Deaths in U.S. 56,000 deaths in U.S. each year Highly treatable if detected early

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Improved Fluoropyrimidine Chemotherapy

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  1. Improved Fluoropyrimidine Chemotherapy William H. Gmeiner, Ph.D. Professor and Chairman Department of Biochemistry, WFUSM

  2. Colon Cancer: 2nd Leading Cause of Cancer Deaths in U.S. • 56,000 deaths in U.S. each year • Highly treatable if detected early • Poor prognosis if diagnosed at late stages • Treatment: Surgery, Radiation and/or Chemotherapy • Most frequently used chemotherapy: 5-Fluorouracil + Leucovorin (5FU/LV)

  3. Web Sites For Reliable Information About Cancer • National Cancer Institute (www.cancer.gov) • American Cancer Society (www.cancer.org) • American Institute of Cancer Research (www.aicr.org) • Wake Forest University Cancer Center (www.wfubmc.edu/cancer)

  4. Cancer Drug Development • Identify Molecular Target: Must Differ Between Malignant and non-Malignant Cells (e.g. involved in cell division). • Determine which types of tumor are most sensitive • Cell culture studies to investigate mechanism of action • Animal studies to demonstrate efficacy in vivo and to determine PK and PD profile. • Clinical studies: Phase I – Safety; Phase II – Optimal Dose; Phase III – Efficacy.

  5. 5FUra - A Clinically Useful Anticancer Drug For More Than 40 Years

  6. Malignant and Non-Malignant Cells Often Differ in Cell Division

  7. The Cell Cycle: Identifying Drug Targets

  8. TS & RR Are Targets For S-Phase Blocking Anticancer Drugs

  9. Toxicities Associated With 5-FU

  10. FdUMP[N] Serves As A Pro-Drug of FdUMP

  11. Recursion Applied to Factorials 10! = 10(9!) However, 9 factorial is 9 times 8 factorial: 9! = 9(8!) Using the recursive concept: 8! = 8(7!) 7! = 7(6!) 6! = 6(5!) 5! = 5(4!) 4! = 4(3!) 3! = 3(2!) 2! = 2(1) 10! = 10(9(8(7(6(5(4(3(2(1))))))))) recursion n. 1 the act of returning. 2 (Math) the repeated application of a mathematical procedure to a preceding result to generate a sequence of values.

  12. Important Initial Questions About FdUMP[10] • Does it work? (i.e. is FdUMP[10] either cytotoxic or cytostatic?). • Is there selectivity for certain malignancies? • Is it safe? (i.e. can FdUMP[10] be used in vivo?) • Is it efficacious? (i.e. can FdUMP[10] inhibit growth of a human tumor?)

  13. Does FdUMP[10] Work?GI50 Values and Relative Potency in the NCI 60 Cell Line Screen • Drug IC50 Relative • 5FU 2.4 x 10-5 1 • FdU 1.8 x 10-6 13.3 • FdUMP[10] 7.1 x 10-8 338.0

  14. Is FdUMP[10] Selective For Certain Malignancies? • Ovarian, NSCLC especially sensitive to FdUMP[10] • Colorectal less sensitive to FdUMP[10]

  15. FdUMP[10] Efficiently Inhibits TS in HT29 Colon Cancer Cells

  16. Inhibition of TS by FdUMP[10] Results in DNA Damage

  17. COMET Assay of HT29 CellsFdUMP[10] Causes Substantially Greater DNA Damage Than 5FU FdUMP[10] 1 x 10-7 M 5FU 1 x 10-6 M

  18. FdUMP[10] Causes S-Phase Arrest of HT29 Cells

  19. FdUMP[10] Inhibits Proliferation and Induces Apoptosis Apoptotic and Proliferative Activity From 40 HT29 Cells Experiment Apoptotic activity Proliferation Control 0 apoptotic events 111 cell divisions 5FU 4 x 10-6 M 3 apoptotic events 20 cell divisions FdUMP[10] 4 x 10-8 M 20 apoptotic events 2 cell divisions

  20. Is FdUMP[10] Safe? • MTD for FdUMP[10] > 200 mg/kg/dose • MTD for 5-FU ~ 40 mg/kg/dose

  21. FdUMP[10] Inhibits Growth of Human Tumor Xenografts

  22. FdUMP[10] Causes Less Damage to GI-Tract Than 5FU Control FdUMP[10] 5FU FdUMP[10] + 5FU

  23. Pre-Clinical Studies With FdUMP[10]: Current Research Objectives • Maximize cellular uptake and target specifically to tumor cells. • Validate TS-Inhibition and DNA Damage as Mechanisms Responsible For Activity. • Determine the Pathway Responsible For Apoptotic Cell Death • Demonstrate Efficacy Towards TS-Overexpressing, 5FU-Resistant Tumors.

  24. Establishing the Link Between TS Inhibition and DNA Damage • TS Inhibition Results in Elevated dUTP/dTTP Ratios • dUTP Misincorporation Initiates Futile Cycles of DNA Repair (UDG; BER) • Direct FdUTP Misincorporation May Also Be Important • Involvement of Topoisomerase I?

  25. Top I Regulates Topological State of DNA For Transcription & Replication • Circular DNA is supercoiled for compaction • Supercoils relieved for access, regenerated for compaction • Top1 Breaks One Strand, Passes Intact Strand Through Break • Alters Linking Number by +/- 1

  26. Topoisomerase I: Target for Nucleoside Analogs? • Top1 is the cellular target for camptothecins (CPTs). • CPTs Stabilize Top1 Cleavage Complexes • DNA Damage Occurs When Stabilized Cleavage Complexes Interact With Replication or Transcription Apparatus • Do Nucleoside Analogs Affect the Stability of Top1 Cleavage Complexes?

  27. AraC and dFdC: 2’-Deoxycytidine Analogs with Distinct Mechanisms and Spectrums of Activity

  28. Nucleoside Analogs Inhibit Key Enzymes And Are Incorporated Into DNA Polymerase Pausing Top1 Inhibition

  29. Relevance of Top1 Inhibition For Efficacy of dFdC, AraC • Top1 Cleavage Complexes Detected in Human Leukemia CEM Cells Treated With dFdC, AraC • P388/CPT45 Top1-Deficient Cells 5-fold resistant to dFdC, 7-10 fold resistant to AraC

  30. Induction of Top1 Cleavage Complexes In Vitro A – DNA alone B - top1 C - top1, CPT

  31. Site-Specific Effects of AraC and dFdC on Top1 Cleavage • 4-6 Fold Enhancement of Top1 Cleavage Complexes When AraC in +1 Position • AraC Enhancement Primarily Due to Inhibition of Top1-Mediated DNA Religation • 5-7 Fold Enhancement of Top1 Cleavage Complexes When dFdC in +1 Position • dFdC Enhances Formation of Top1 Cleavage Complexes • dFdC Induced One New Top1 Cleavage Site Observed In Presence of CPT

  32. Structure of Top1 Cleavage Complex With AraC in +1 Position of the Non-Scissile Strand

  33. Structural Basis For Effects of AraC and dFdC On Top1 Cleavage • NMR Structures of AraC- and dFdC-Substituted Model Okazaki Fragments • NMR Studies of 31mer DNA Hairpin Containing a Single Top1 Cleavage Site • Preparation of 31mer DNA Hairpin With dFdC and AraC Substitution

  34. AraC-, dFdC-Substituted Model Okazaki Fragments

  35. NMR Structures of dFdC-, and AraC-Substituted Model Okazaki Fragments

  36. AraC-, dFdC-Substitution Destabilizes Duplex Structure • [OKA] Tm 46.8 oC • [AraC] Tm 42.4 oC • [GEM] Tm 41.2 oC

  37. AraC Sugar Pucker Differs From dC in Model Okazaki Fragment P ~ 110o P ~ 160o

  38. AraC Sugar Pucker Disrupts Base Stacking in Adjacent Strand

  39. Structural Effects of AraC:Implications For Top1 Cleavage • AraC Adopts C2’-endo Sugar Pucker • Stereoelectronic Effects Cause Arabinosyl Sugar to be More Rigid Than 2’-Deoxyribose • Rigidity of Arabinosyl Sugar May Inhibit Religation of Top1 Cleavage Complexes

  40. dFdC Adopts C3’-endo Sugar Pucker With Altered Electrostatic Surface Relative to dC P ~ 30o P ~ 110o

  41. Electrostatic Surface of dFdC and dC in [GEM] and [OKA]

  42. Structural Effects of dFdC:Implications For Top1 Cleavage • dFdC Adopts C3’-endo sugar pucker with altered electrostatic surface relative to dC • Electronegativity of Fluorines in dFdC may contribute to Enhanced Formation of Top1 Cleavage Complexes • Relative Flexibility of dFdC Allows Facile Re-Ligation to Occur

  43. Top1: Target for FdUMP[10]?

  44. Conclusions • FdUMP[10] Is a Prototype of a New Fluoropyrimidine That Is Safer and More Effective Than 5FU • FdUMP[10] Blocks HT29 Cells in S-Phase and Causes DNA Damage • Top1 May Be Involved in Mediating Damage Resulting From FdUMP[10] Exposure

  45. Acknowledgements • Current Group: Cui Wei, Xi-an Mao, Debbie Boles. • Former Group: Jinqian Liu, Changnian Liu, Parag Sahasrabudhe. • Top1: Yves Pommier (NCI); Phillipe Pourquier (Bordeaux). • Structural Biology: Tom James (UCSF); Dave Konerding UCSF. • Funding: WFUSM; CCCWFU

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