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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.

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Cyclopamine and the Hedgehog Signaling Pathway

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  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

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