1 / 59

Cyclopamine and the Hedgehog Signaling Pathway

Cyclopamine and the Hedgehog Signaling Pathway. Marc Legault Department of Chemistry University of Ottawa November 5 th , 2009. Cyclopamine and the Hedgehog Signaling Pathway. ABC- nor- D- homo Spiro tetrahydrofuran E ring Piperidine F ring.

trevor
Download Presentation

Cyclopamine and the Hedgehog Signaling Pathway

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Cyclopamine and the Hedgehog Signaling Pathway Marc Legault Department of Chemistry University of Ottawa November 5th, 2009

  2. Cyclopamine and the Hedgehog Signaling Pathway • ABC-nor-D-homo • Spiro tetrahydrofuran E ring • Piperidine F ring Polyphemus, Johann Heinrich Wilhelm Tischbein, 1802 J. D. Winkler, A. Isaacs, L. Holderbaum, V. Tatard, and N. Dahmane, Org. Lett. 2009, 11, 2824-2827

  3. Cyclopamine and cyclopia • Holoprosencephaly(cyclopia) common phenotype • found in livestock(1957-1968) Veratrum californicum Corn lily Lamb afflicted with cyclopia as a result of cyclopamine ingestion W. Binns, L.F James, R.F. Keeler, and L.D. Balls, Cancer Res. 1968, 28, 2323-2326 J. D. Winkler, A. Isaacs, L. Holderbaum, V. Tatard, and N. Dahmane, Org. Lett. 2009, 11, 2824-2827

  4. Causative agents • Seminal work performed by Richard F. Keeler and Wayne Binns • Isolated, characterized, and tested effects of 18 different compounds • Jervine & cyclopamine found to be teratogenic The skulls of a cyclopic and normal sheep R.F. Keeler, W. Binns, Teratology1968, 1, 5-10

  5. Pathway discovery • Drosophila melanogaster larvae screens • Identified 15 loci involved in segmental patterning during development • Hedgehog: vertebrate homologues: Desert, Indian, Sonic C. Nüsslein-Volhard, & E. Wieschaus. Nature 1980,287, 795-801

  6. Pathway discovery • Drosophila melanogaster Heidelberg screens • Nobel prize in medicine/physiology (1995) Eric Wieschaus Princeton Christiane Nüsslein-Volhard Max Planck institute for Developmental Biology Edward B. Lewis 1918-2004 C. Nüsslein-Volhard, & E. Wieschaus. Nature 1980,287, 795-801

  7. Pathway explained • In the absence of ligand, Ptc inhibits Smo N. Mahindroo, C. Punchihewa, N Fujii, J. Med. Chem. 2009, 52, 3829-3845

  8. Pathway explained • Activated hedgehog interacts with Ptc • Hedgehog (Shh) relieves Ptc inhibition of Smo L. Jacob & L. Lum, Science2007, 317, 66-68 N. Mahindroo, C. Punchihewa, N Fujii, J. Med. Chem. 2009, 52, 3829-3845

  9. Pathway explained • Downstream activation of • GLi zinc finger transcription factors L. Jacob & L. Lum, Science2007, 317, 66-68 N. Mahindroo, C. Punchihewa, N Fujii, J. Med. Chem. 2009, 52, 3829-3845

  10. Pathway explained • Upregulation of • target genes L. Jacob & L. Lum, Science2007, 317, 66-68 N. Mahindroo, C. Punchihewa, N Fujii, J. Med. Chem. 2009, 52, 3829-3845

  11. Development • Brain, lungs, skeletal, musculature, GI tract development • Misregulation of the pathway can have serious developmental implications i.e. cyclopia *C.J. Tabin et al. Cell 2004,118, 517-528 *C.J. Tabin & A.P. McMahon, Science2008, 321, 350-352

  12. Cyclopamine interaction • Synthesis of photo-activated isotopic cross-linking derivative *J.K. Chen, J. Taipale, M.K. Cooper, P.A. Beachy, Gene. Dev.2002, 16, 2743-2748

  13. Cyclopamine interaction • BODIPY based fluorescent probe showed colocalization with Smo *J.K. Chen, J. Taipale, M.K. Cooper, P.A. Beachy, Gene. Dev.2002, 16, 2743-2748

  14. Pathway & cyclopamine L. Jacob & L. Lum, Science2007, 317, 66-68 N. Mahindroo, C. Punchihewa, N Fujii, J. Med. Chem. 2009, 52, 3829-3845

  15. “...nothing is evil which is according to nature.” -Marcus Aurelius Antonius

  16. Hedgehog and cancer • Hh pathway also active in stem cells • Many types of cancer are believed to arise through stem cell pathway J. Taipale, P.A. Beachy, Nature2001, 411, 349-354 *P.A. Beachy, S.S. Karhadkar, D.M. Berman, Nature 2004, 432, 324-332 P.A. Beachy et al. Proc. Natl. Acad. Sci. USA2007, 104, 4048-4053

  17. Cancer therapy • Lung, liver, stomach, prostate, pancreatic and basal cell carcinoma • Selective targeting of the aberrant pathway in cells could serve as a potential chemotherapy N. Mahindroo, C. Punchihewa, N Fujii, J. Med. Chem. 2009, 52, 3829-3845

  18. Cancer statistics Cancer Research UK <http://cancerresearchuk.org>

  19. Issues with a cyclopamine drug • Chemical • Solubility • Sensitivity • Biological • Cognitive effects • Stem cell pool depletion • $$ J. D. Winkler, A. Isaacs, L. Holderbaum, V. Tatard, and N. Dahmane, Org. Lett. 2009, 11, 2824-2827 P.A. Beachy, S.S. Karhadkar, D.M. Berman, Nature 2004, 432, 324-332

  20. Formal Synthesis(retrosynthesis) • Extremely laborious • Very low yielding • Isolation has been used to obtain quantities of cyclopamine T. Masamune et al. Bull. Chem. Soc. Jpn. 1965,38, 1374- 1378 T. Masamune et al. J. Am. Chem. Soc. 1967, 89, 4521-4523 W.S. Johnson et al. J. Am. Chem. Soc. 1967, 89, 4523-4524 W.S. Johnson et al. J. Am. Chem. Soc. 1967, 89, 4524-4526

  21. Semi-synthesis highlights • Biomimetic hydroxylation • Wagner-Meerwein type ring expansion • Access to unnatural isomers A. Giannis et al. Angew. Chem. Int. Ed. 2009, 48, 7911-7914

  22. Retrosynthesis of cyclopamine A. Giannis et al. Angew. Chem. Int. Ed. 2009, 48, 7911-7914

  23. Semi-synthesis:ABCD framework • How to introduce hydroxyl group at C-12 position for • subsequent rearrangement? A. Giannis et al. Angew. Chem. Int. Ed. 2009, 48, 7911-7914

  24. Biomimetic hydroxylation W. Kaim, J. Rall, Angew. Chem. Int. Ed. Eng. 1996, 35, 43-60 L. M. Mirica, X. Ottenwaelder, and D.P. Stack , Chem. Rev. 2004, 104, 1013-1045

  25. Non-activated CH2 hydroxylations β-Hydroxylation of D ring β-Hydroxylation of D-homo ring γ-Hydroxylation of C ring B. Schönecker et al.Angew. Chem. Int. Ed. 2003, 42, 3240-3244 A. Magyar et al.Tetrahedron: Asymmetry, 2003, 14, 1925-1934 B. Schönecker et al. Chem. Eur. J. 2004, 10, 6029-6042

  26. Non-activated CH2hydroxylations • Oxygen can react with hydrogen on β face. Model for Copper dioxygen complex B. Schönecker et al. Chem. Eur. J. 2004, 10, 6029-6042

  27. Proposed mechanism • First proposed by Fukuzumi et al. Bis(μ-oxo) complex μ-η²:η²-peroxo complex S. Fukuzumi et al.Angew. Chem. Int. Ed. 2000, 39, 398-400 P. Spuhler & M.C. Holthausen, Angew. Chem. Int. Ed. 2003, 42, 5961-5965 B. Schönecker et al. Chem. Eur. J. 2004, 10, 6029-6042

  28. Semi-synthesis:ABCD framework A. Giannis et al. Angew. Chem. Int. Ed. 2009, 48, 7911-7914 K.W.C. Poon, & G.B. Dudley, J. Org. Chem. 2006, 71, 3923-3927 B. Schönecker et al.Angew. Chem. Int. Ed. 2003, 42, 3240-3244

  29. Semi-synthesis:E ring • Two possible routes to lead to (natural) 20S diastereomer • Initial route to (natural) 20S diastereomer will be described first • A. Giannis et al. Angew. Chem. Int. Ed. 2009, 48, 7911-7914 • C.W. Shoppee, Nature1950, 166, 107-108 • H. Van Bekkum, Synthesis 1996 (10) 1153-1173

  30. Semi-synthesis:E Ring • 54% over six steps to obtain E ring with proper stereochemistry • A more direct route was found A. Giannis et al. Angew. Chem. Int. Ed. 2009, 48, 7911-7914

  31. Alternative route to lactone • More concise route to 20S diastereomer • Provides unnatural diastereomer as well 6:4 diastereomeric ratio A. Giannis et al. Angew. Chem. Int. Ed. 2009, 48, 7911-7914 H.C. Brown, E.F. Knights, & C.G. Scouten, J. Am. Chem. Soc. 1974, 96, 7765-7770 H. Van Bekkum, Synthesis 1996 (10) 1153-1173

  32. Semi-synthesis:ABC-nor-D-homo • Triflic anhydride promoted Wagner-Meerwein type rearrangement • 3:7 internal/external regioisomeric ratio A. Giannis et al. Angew. Chem. Int. Ed. 2009, 48, 7911-7914 T. Shono, K. Fujita, & S. Kumai, Tetrahedron Lett.1973, 14, 3123-3126

  33. Semi-synthesis:F ring A. Giannis et al. Angew. Chem. Int. Ed. 2009, 48, 7911-7914 D.A. Evans, T.C. Britton, J. Am. Chem. Soc. 1987, 109, 6881-6883

  34. Semi-synthesis:F ring A. Giannis et al. Angew. Chem. Int. Ed. 2009, 48, 7911-7914 D.A. Evans, T.C. Britton, J. Am. Chem. Soc. 1987, 109, 6881-6883

  35. Semi-synthesis:F ring A. Giannis et al. Angew. Chem. Int. Ed. 2009, 48, 7911-7914 T. Imamato et al.J. Org. Chem. 1984, 49, 3904-3912 H. Staudinger, J. Meyer, Helv. Chim. Acta 1919, 2, 635-646 C.R. Johnson & B.D. Tait, J. Org. Chem. 1987, 52, 281-283

  36. Semi-synthesis:F ring 3 : 1 A. Giannis et al. Angew. Chem. Int. Ed. 2009, 48, 7911-7914

  37. Semi-synthesis:F Ring G. Deleris, J. Kowalski, J. Dunogues, & R. Calas, Tetrahedron Lett.1977, 18, 4211-4214 A. Giannis et al. Angew. Chem. Int. Ed. 2009, 48, 7911-7914

  38. Synthesis overview • 20 steps • Key steps: • Biomimetic hydroxylation • Wagner-Meerwein ring expansion • Access to unnatural analogs • 1% overall yield A. Giannis et al. Angew. Chem. Int. Ed. 2009, 48, 7911-7914

  39. Clinical studies • Topical cream • Induces apoptosis • Treatment before/after • One of several drugs in clinical trials S. Tabs & O. Avci. Eur. J. Dermatol. 2004, 14, 96-102

  40. Issues with a cyclopamine drug • Chemical • Solubility • Sensitivity • Biological • Cognitive effects • Stem cell pool depletion • $$ J. D. Winkler, A. Isaacs, L. Holderbaum, V. Tatard, and N. Dahmane, Org. Lett. 2009, 11, 2824-2827 P.A. Beachy, S.S. Karhadkar, D.M. Berman, Nature 2004, 432, 324-332

  41. Cheaper analogs • Energy minimization calculations led to the identification of homologous structures • Relative ease of synthesis of one over the other J. D. Winkler, A. Isaacs, L. Holderbaum, V. Tatard, and N. Dahmane, Org. Lett. 2009, 11, 2824-2827

  42. Synthesis of estrone derived analog • 4 steps, 50.5 % overall yield • Commercially available starting material • Spiro ring and nitrogen orientation most important for activity J. D. Winkler, A. Isaacs, L. Holderbaum, V. Tatard, and N. Dahmane, Org. Lett. 2009, 11, 2824-2827

  43. Biological assay • μM inhibitor of Shh signaling J. D. Winkler, A. Isaacs, L. Holderbaum, V. Tatard, and N. Dahmane, Org. Lett. 2009, 11, 2824-2827

  44. Issues with a cyclopamine drug • Chemical • Solubility • Sensitivity • Biological • Cognitive effects • Stem cell pool depletion • $$ J. D. Winkler, A. Isaacs, L. Holderbaum, V. Tatard, and N. Dahmane, Org. Lett. 2009, 11, 2824-2827 P.A. Beachy, S.S. Karhadkar, D.M. Berman, Nature, 2004, 432, 324-332

  45. Improving localized activity • Strategy: Improve targeting specificity to cancerous cells • Mu-SSKYU peptide chain linked to cyclopamine • Cleaved to active drug by prostate specific antigen (PSA) S.K. Kumar et al.Bioorg. Med. Chem. 2008, 16, 2764-2768

  46. Improving localized activity • Half life of prodrug 3 h • PSA dependence on activity S.K. Kumar et al.Bioorg. Med. Chem. 2008, 16, 2764-2768

  47. Issues with a cyclopamine drug • Chemical • Solubility • Sensitivity • Biological • Cognitive effects • Stem cell pool depletion • $$ J. D. Winkler, A. Isaacs, L. Holderbaum, V. Tatard, and N. Dahmane, Org. Lett. 2009, 11, 2824-2827 P.A. Beachy, S.S. Karhadkar, D.M. Berman, Nature 2004, 432, 324-332

  48. Decreasing sensitivity • Strategy: retain steroidal structure even in the presence of acid M.R. Tremblay et al.J. Med. Chem. 2008, 51, 6646-6649

  49. Decreasing sensitivity M.R. Tremblay et al.J. Med. Chem. 2008, 51, 6646-6649

  50. Improving metabolic stability & potency • Oppenauer oxidation and migration of double bond furnished a more soluble inhibitor • Further improvements to enhance metabolic stability and potency M.R. Tremblay et al.J. Med. Chem. 2008, 51, 6646-6649 M.R. Tremblay et al. J. Med. Chem. 2009, 52, 4400-4418

More Related