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Presented By : HARDIK PARIKH Department of Medicinal Chemistry

For more presentations and information visit http://www.pharmaxchange.info. CDC25 PHOSPHATASE: A POTENTIAL TARGET FOR NOVEL ANTICANCER AGENTS. Presented By : HARDIK PARIKH Department of Medicinal Chemistry Institute for Structural Biology and Drug Discovery

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Presented By : HARDIK PARIKH Department of Medicinal Chemistry

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  1. For more presentations and information visit http://www.pharmaxchange.info CDC25 PHOSPHATASE: A POTENTIAL TARGET FOR NOVEL ANTICANCER AGENTS Presented By: HARDIK PARIKH Department of Medicinal Chemistry Institute for Structural Biology and Drug Discovery Virginia Commonwealth University email: parikhhi@mymail.vcu.edu 30/10/2009

  2. For more presentations and information visit http://www.pharmaxchange.info Outline • Timeline of Cancer • Cell Cycle Regulation of Cdc25 Phosphatases • Structure of Cdc25 Phosphatases • Catalytic Mechanism of Cdc25 Phosphatases • Small Molecule Inhibitors of Cdc25 Phosphatases • Future Prospects 2

  3. For more presentations and information visit http://www.pharmaxchange.info What is Cancer? • According to NCI, • “Cancer is a term used for diseases in which abnormal cells divide without control and are able to invade other tissues.” 3 NCI Website - http://www.cancer.gov/cancertopics/what-is-cancer

  4. For more presentations and information visit http://www.pharmaxchange.info Timeline of Cancer • 3000BC: Earliest observations of cancer • Bone remains of mummies have • revealed growths suggestive of the bone cancer. • The Edwin Smith Papyrus, oldest descriptions • of cancer known, described 8 cases of tumors. • Origin of word Cancer • Credited to Greek physician Hippocrates (460-370 BC). He used the terms ‘carcinos’ and ‘carcinoma’. 4 ACS Website- http://www.cancer.org/docroot/CRI/content/CRI_2_6x_the_history_of_cancer_72.asp Images adapted from – http://www.cancerquest.org (accessed 10/22/09)

  5. For more presentations and information visit http://www.pharmaxchange.info Timeline of Cancer • 1761: GiovanniMorgagniofPaduawas the first to perform autopsies to relate the patient's illness to the pathologic findings after death. • 1890 : First Cancer Treatment • William Halsted, the first professor of surgery at John Hopkins, Harvard, and Yale, performed the first radical mastectomy. • 1914: Mutation theory of cancer • Theodor Boveri proposed the Somatic Mutation Theory of Cancer. He believed that cancer was caused by abnormal chromosomes. ACS Website- http://www.cancer.org/docroot/CRI/content/CRI_2_6x_the_history_of_cancer_72.asp Images adapted from – http://www.cancerquest.org (accessed 10/22/09) 5

  6. For more presentations and information visit http://www.pharmaxchange.info Timeline of Cancer • 1940s: Era of Cancer Chemotherapy • Goodman and Gilman suggested that • nitrogen mustards could be used to treat lymphoma. • 1971: War on Cancer declared by President Nixon • The National Cancer Act was signed into law; additional $100 million funds released to find a cure for cancer. • 2003: Human Genome Project • Identified ~25,000 genes in human DNA. • 2006: First cancer vaccine • FDA approved Gardasil, a vaccine that protects against HPV – Human papillomavirus, major cause for cervical cancer. ACS Website- http://www.cancer.org/docroot/CRI/content/CRI_2_6x_the_history_of_cancer_72.asp Images adapted from – http://www.cancerquest.org (accessed 10/22/09) 6

  7. For more presentations and information visit http://www.pharmaxchange.info Current Scenario • Cancer – the second leading cause of deaths worldwide. • WHO has estimated 12 million deaths due to cancer worldwide in 2030. • According to American Cancer Society, • About 1.5 million new cancer cases and more than 500,000 deaths are expected in USA alone in 2009. • Half of all men and one-third of all • women in the United States will • develop cancer during their lifetimes. • Cancer is the reason of 1 out of • every 4 deaths in USA. CANCER WHO website - http://www.who.int/mediacentre/factsheets/fs297/en/index.html (accessed 10/22/09). Jemal, A. et al. CA Cancer J Clin. 2009, 59, 225-249. 7

  8. For more presentations and information visit http://www.pharmaxchange.info Targeting Cancer All cancers share a common feature – rapid and uncontrolled cell proliferation. Normal Cell Cycle Cancerous Cell Cycle Cdk M M Cyc G0 G0 G2 G2 G1 G1 Regulated activity Misregulation/ Over activation S S Cdc25 Phosphatase CANCER Activates Cell Cycle Regulator 8

  9. For more presentations and information visit http://www.pharmaxchange.info Cell Division Cycle 25 (Cdc25) Phosphatase • Control the progression of cell cycle through activating Cyclin-dependent Kinase(Cdk) – Cyclin complexes • In the event of DNA damage – • Key targets of the checkpoint machinery that ensures genetic stability • They are Dual Specificity Phosphatases (DSP), a subfamily of Protein Tyrosine Phosphatases (PTPs). 9 Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.

  10. For more presentations and information visit http://www.pharmaxchange.info Cdc25 Isoforms In mammalian cells,threeIsoforms have been identified : Cdc25A, Cdc25B, Cdc25C 10 Boutros, R. et al. Nature Reviews Cancer, 2007, 7, 495-507.

  11. For more presentations and information visit http://www.pharmaxchange.info Cell Cycle Regulation by Cdc25 Phosphatases 11

  12. For more presentations and information visit http://www.pharmaxchange.info Activation of the Cdk/cyclin complex Cell cycle progression requires activation of the cyclin-dependent kinases(Cdk). Cdk Cyclin-Dependent Kinase Cyclin Myt1/Wee1 Cyc CAK Cdk Activating Kinase CAK CDC25 Phosphorylation Dephosphorylation T161 Cdk T14 Y15 p T161 p p Cdk p Cdk Cyc Cdk Cyc Cyc (Active) (Inactive) p p Cyc 12 Ducommun, B. et al. Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 818-824.

  13. For more presentations and information visit http://www.pharmaxchange.info • Regulation of Cell Cycle Transition • Different isoforms activate different complexes - • Cdc25A mainly controls the G1/S Transitions via the dephosphorylation • and activation of the • Cdk2/CyclinE and • Cdk2/CyclinA • complexes. Cdc25B Cdk2 Cdk1 Cdk1 Cdk2 Cdc25C • Cdc25B • activates • Cdk1-CyclinB • at the centrosome • during the G2/M • transition. • Cdc25C activates • The Cdk1-CyclinB • complex in the nucleus • at the onset of mitosis. CycA CycE CycB CycB M G1 G2 S Cdc25A 13 Boutros, R. et al. Nature Reviews Cancer, 2007, 7, 495-507.

  14. For more presentations and information visit http://www.pharmaxchange.info The Checkpoint Response DNA Damage Checkpoint Kinase 1 Checkpoint Kinase 2 Mitogen-activated Protein Kinase Activated Protein Kinase 2 / MAPKAP Kinase2 Cdc25 Phosphatases Degradation via proteosome 14 Ducommun, B. et al. Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 818-824.

  15. For more presentations and information visit http://www.pharmaxchange.info The Checkpoint Response DNA Damage DNA Damage DNA Damage Degradation via proteosome Cytoplasmic Sequestration Cytoplasmic Sequestration 15 Ducommun, B. et al. Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 818-824.

  16. For more presentations and information visit http://www.pharmaxchange.info • The Checkpoint Response • Cell cycle arrest - Cdc25B Cdk1 Cdk2 Cdk2 Cdk1 Cdc25C CycA CycE CycB CycB M G1 G2 S Cdc25A 16 Ducommun, B. et al. Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 818-824.

  17. For more presentations and information visit http://www.pharmaxchange.info Cdc25 overexpressioncauses Tumors • Over-activation of Cdk-cyclin complexes – pushes cell cycle in untimely manner. Cdk2 Cdk2 Cdk1 Cdk1 Cdc25A overexpression accelerates entry into S-phase Cdc25B CycB CycB CycE CycA M Cdc25C G1 Cdc25B over- expression rapidly pushes the S or G2 phase cells into mitosis even with incompletely replicated DNA. G2 S Cdc25A 17 Boutros, R. et al. Nature Reviews Cancer, 2007, 7, 495-507.

  18. For more presentations and information visit http://www.pharmaxchange.info Cdc25 overexpression: A recurring theme in Cancer 18 Boutros, R. et al. Nature Reviews Cancer, 2007, 7, 495-507.

  19. For more presentations and information visit http://www.pharmaxchange.info Structure of Cdc25 Phosphatases 19

  20. For more presentations and information visit http://www.pharmaxchange.info Structure C-terminal region Catalytic Domain N-terminal region Regulatory Domain • N-terminal regions are highly divergent • Contains sites for • phosphorylation • ubiquitination • which regulate phosphatase • activity. • Contains signals to control the intracellular localization • C-terminal regions are highly homologous • (~60% pairwise identity over ~200 amino acids) • Contains the Catalytic Site • The HCX5R motif • His – Cys – XXXXX – Arg • conserved within the PTP family 20 Rudolph, J. Biochemistry, 2007, 46, 3595-3604.

  21. For more presentations and information visit http://www.pharmaxchange.info Structure Cdc25A (PDB ID: 1c25) Cdc25B (PDB ID: 1qb0) 21

  22. For more presentations and information visit http://www.pharmaxchange.info Crystal Structure of Catalytic Domain of Cdc25B • Red – Active site • loop (HCX5R) • Blue - Sulfate Top-view Side-view (PDB ID : 1qb0) 22

  23. For more presentations and information visit http://www.pharmaxchange.info Crystal Structure of Catalytic Domain of Cdc25B • Crystal structure of the catalytic domain of Cdc25B was solved by X-ray Crystallography at 1.9Å resolution. • The active site loop contains the signature HCX5R sequence. • Red – Active site • loop (HCX5R) • Blue - Sulfate Top-view 23 Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.

  24. For more presentations and information visit http://www.pharmaxchange.info Crystal Structure of Catalytic Domain of Cdc25B • HCX5R motif • Histidine 472 H • Cysteine 473 C • Glutamic acid 474 • Phenylalanine 475 • Serine 476 X • Serine 477 • Glutamic acid 478 • Arginine 479 R • Backbone amides of five X resides along with arginine form multiple H-bonds with the bound sulfate. 24 Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.

  25. For more presentations and information visit http://www.pharmaxchange.info Crystal Structure of Catalytic Domain of Cdc25B • HCX5R motif • Histidine 472 H • Cysteine 473 C • Glutamic acid 474 • Phenylalanine 475 • Serine 476 X • Serine 477 • Glutamic acid 478 • Arginine 479 R • The thiolate anion of cysteine lies directly below the bound sulfate. 25 Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.

  26. For more presentations and information visit http://www.pharmaxchange.info Crystal Structure of Catalytic Domain of Cdc25B Active site • The active site pocket is small and extremely shallow. • Gets filled up completely by the phosphoryl group of the substrate alone. • Allows access to both pThr and pTyr containing substrates, in accord with its dual-specificity nature. 26 Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.

  27. For more presentations and information visit http://www.pharmaxchange.info Crystal Structure of Catalytic Domain of Cdc25B • A large cavity adjacent to the catalytic pocket was identified • Called “swimming-pool” for the abundance of well ordered water molecules Active site Yellow – Active site cysteine Red – Water molecules Swimming Pool 27 Rudolph, J. Mol Pharmacol. 2004, 66, 780-782.

  28. For more presentations and information visit http://www.pharmaxchange.info Crystal Structure of Catalytic Domain of Cdc25B Catalytic pocket Swimming pool 28 Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.

  29. For more presentations and information visit http://www.pharmaxchange.info Catalytic Mechanism of Cdc25 Phosphatases 29

  30. For more presentations and information visit http://www.pharmaxchange.info • Catalytic Mechanism • Reaction mechanism for PTPs - 30 Chen, W. et al. Biochemistry, 2000, 39, 10781-10789.

  31. For more presentations and information visit http://www.pharmaxchange.info Catalytic Mechanism • Identity of catalytic acid – • No sequence conservation with other PTPs • Asp383 of Cdc25A was implicated as catalytic acid on the basis of reduction of activity of D383N mutant. • Glu474 of Cdc25B (corresponding to Glu431 in Cdc25A), the first of the five X residues, could serve the role of the catalytic acid. • Glu478 of Cdc25B (corresponding to Glu435 in Cdc25A), the last of the five X residues, is a more likely candidate for the catalytic acid. 31 Chen, W. et al. Biochemistry, 2000, 39, 10781-10789.

  32. For more presentations and information visit http://www.pharmaxchange.info • Catalytic Mechanism • Enzyme uses a monoprotonatedsubstrate • The protein might use as its substrate a monoprotonated phosphate in contrast to the typical bisanionic phosphate, because of higher intrinsic reactivity. 32 Rudolph, J. et al. Biochemistry, 2002, 41, 14613-14623.

  33. For more presentations and information visit http://www.pharmaxchange.info • Catalytic Mechanism • Enzyme uses a monoprotonated substrate 33 Rudolph, J. et al. Biochemistry, 2002, 41, 14613-14623.

  34. For more presentations and information visit http://www.pharmaxchange.info Small Molecule Inhibitors of Cdc25 Phosphatases 34

  35. For more presentations and information visit http://www.pharmaxchange.info Potential Druggable Targets for Cdc25 Enzyme Activity Transcription Translation Cdc25 Post – Translation Degradation Subcellular Localization Protein-Protein Interaction 35 Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.

  36. For more presentations and information visit http://www.pharmaxchange.info Inhibitors of Cdc25 • Natural products • Lipophilic acids • Quinones • Electrophiles • Sulfonylatedaminothiazoles • Phosphate mimics 36 Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.

  37. For more presentations and information visit http://www.pharmaxchange.info Inhibitors of Cdc25 • Natural products • Lipophilic acids • Quinones • Electrophiles • Sulfonylatedaminothiazoles • Phosphate mimics 36 Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.

  38. For more presentations and information visit http://www.pharmaxchange.info Inhibitors of Cdc25 • Natural products • Lipophilic acids • Quinones • Electrophiles • Sulfonylatedaminothiazoles • Phosphate mimics 36 Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.

  39. For more presentations and information visit http://www.pharmaxchange.info Inhibitors of Cdc25 • Natural products • Lipophilic acids • Quinones • Electrophiles • Sulfonylatedaminothiazoles • Phosphate mimics 36 Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.

  40. For more presentations and information visit http://www.pharmaxchange.info Inhibitors of Cdc25 • Natural products • Lipophilic acids • Quinoids • Electrophiles • Sulfonylatedaminothiazoles • Phosphate mimics 36 Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.

  41. For more presentations and information visit http://www.pharmaxchange.info Inhibitors of Cdc25 • Natural products • Lipophilic acids • Quinoids • Electrophiles • Sulfonylatedaminothiazoles • Phosphate mimics 36 Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.

  42. For more presentations and information visit http://www.pharmaxchange.info Inhibitors of Cdc25 • Natural products • Lipophilic acids • Quinoids • Electrophiles • Sulfonylatedaminothiazoles • Phosphate mimics 36 Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.

  43. For more presentations and information visit http://www.pharmaxchange.info Inhibitors of Cdc25 • Natural products • Lipophilic acids • Quinones as Inhibitors of Cdc25B • Electrophiles • Sulfonylatedaminothiazoles • Phosphate mimics 36 Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.

  44. For more presentations and information visit http://www.pharmaxchange.info Quinones as Inhibitors of Cdc25B • Electrophilic properties of quinones suggest two possbile interactions with enzyme : • a sulfhydrylarylation of cysteine • an ether linkage of serine • Can also oxidize the catalytic thiolate group of Cys473 37 Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.

  45. For more presentations and information visit http://www.pharmaxchange.info Quinones as Inhibitors of Cdc25B Benzothiazole/ Benzoxazole – diones Naphthoquinones Quinolinediones Indolyldihydroxy-quinone 38 Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.

  46. For more presentations and information visit http://www.pharmaxchange.info Quinones as Inhibitors of Cdc25B • Naphthoquinones Covalently inhibits enzyme by arylating the catalytic cysteine IC50 = 0.125 μM* NSC672121 IC50 = 3.8 μM* NSC95397 * in-vitro IC50 values 39 Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.

  47. For more presentations and information visit http://www.pharmaxchange.info Quinones as Inhibitors of Cdc25B • Naphthoquinones IC50 = 0.125 μM* IC50 = 4.13 μM* IC50 = 3.8 μM* IC50 = 1.75 μM* * in-vitro IC50 values 40 Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.

  48. For more presentations and information visit http://www.pharmaxchange.info Quinones as Inhibitors of Cdc25B • Naphthoquinones IC50 = 10.3 μM* IC50 = 4.1 μM* IC50 = 12.9 μM* IC50 = 1.8 μM* * Growth inhibitory IC50 values for MCF7 human breast cancer cell lines 41 Peyregne, V. P. et al. Mol. CacncerTher., 2005, 4, 595-602.

  49. For more presentations and information visit http://www.pharmaxchange.info Quinones as Inhibitors of Cdc25B • Naphthoquinones Hydrogen bonding between the enolic anion and the hydroxy group 42 Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.

  50. For more presentations and information visit http://www.pharmaxchange.info Quinones as Inhibitors of Cdc25B • Naphthoquinones • Binding Mode NSC 128981 IC50 = 0.62 μM Result of 50 independent Autodock and GOLD docking runs – 43 Lavechhia, A. et al. Chem Med Chem, 2006,1, 540-550.

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