Geldanamycin: A Novel Approach to Treating Cancer

1
Geldanamycin A Novel Approach to Treating Cancer

• Elisabeth von Moos
• December 1, 2005

2
What is Cancer?

• More than 100 different and distinct diseases.

Group of diseases characterized by the
uncontrolled growth of cells or the failure of
cells to die normally.

• An estimated 149,000 new cases of cancer will be
  
• diagnosed in Canada in 2005.
• 38 of Canadian women and 44 of Canadian men
• will develop cancer during their lifetimes.

3
Current Cancer Therapies
Surgery Radiation therapy Biological therapy
Photodynamic therapy
Many cancers have no cure. Poor prognosis for
patients with solid tumors.
Chemotherapy
Targeted cancer therapy
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Important Therapeutic Targets
Steroid receptors
PR, AR, GCR
V-Src Akt Raf-1 Bcr-Abl
Mutant p53 HIF-1a
Therapy
Soluble kinases
Transcription factors
ErbB2
Transmembrane kinases
Neckers, N. Trends Mol. Med. 2002, 8, S55.
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Heat Shock Protein 90
Steroid receptors
PR, AR, GCR
V-Src Akt Raf-1 Bcr-Abl
Mutant p53 HIF-1a
Hsp90
Soluble kinases
Transcription factors
ErbB2
Transmembrane kinases
Neckers, N. Trends Mol. Med. 2002, 8, S55.
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Heat Shock Proteins
Degrade denatured proteins
Repair denatured proteins
Prevent protein aggregation
Intracellular protein transport
Fold nascent proteins or repair misfolded
proteins
Control regulatory proteins
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Central Dogma of Molecular Biology
DNA RNA
Transcription Translation
Protein
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Molecular Chaperones
nascent protein
mature protein
ATP ADP Pi
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Heat Shock Proteins Protect Cells
stress
Stress denatures proteins
Denatured proteins detected
Heat shock proteins produced
Denatured proteins refolded
10
Heat Shock Protein 90

• One of the most abundant cellular proteins
• Functions in a multi-component complex
• Captures and holds client proteins in
  intermediate states of folding
• ATP-dependant

ATP
Prodromou, C. Nature Struct. Biol. 1997, 4, 477.
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Heat Shock Protein 90

• One of the most abundant cellular proteins
• Functions in a multi-component complex
• Captures and holds client proteins in
  intermediate states of folding
• ATP-dependant

ADP
Prodromou, C. Nature Struct. Biol. 1997, 4, 477.
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Mechanism of Action of Hsp90
unfolded
ImmunoPs
Cyp40
client
p50Cdc37
p23
ATP hydrolysis
ATP exchange
ADP
ATP
Hsp40
Hip
PP5
p60Hop
Hsp70
client
BAG-1
folded
Proteosome
Isaacs, J.S. Cancer Cell, 2003, 3, 213.
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The Ansamycin Antibiotics
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Geldanamycin

• Isolated in 1970 fromStreptomyces hygroscopicus
• Shows anti-microbial, anti-viral, and anti-tumor
  activity

DeBoer, C. et al. J. Antibiot. 1970, 33, 781.
Merrell, P.H. et al. J.
Am. Chem. Soc. 1970, 92, 7591.
15
Macbecin 1

• Isolated in 1980 from Nocardia
• Shows anti-microbial, anti-viral, and anti-tumor
  activity

Tanida, S. et al. J. Antibiot. 1980, 33, 199.
Muroi, M. et al.
Tetrahedron, 1981, 37, 1123.
16
Herbimycin A

• Isolated in 1980 from Nocardia
• Shows anti-microbial, anti-viral, and anti-tumor
  activity

Tanida, S. et al. J. Antibiot. 1980, 33, 199.
Muroi, M. et
al.Tetrahedron, 1981, 37, 1123.
17
Herbimycin A Tatsuta Synthesis
16
15
15
3
4
4
Tatsuta, K. et al. Tetrahedron Lett. 1991, 32,
6015.
18
Herbimycin A Martin Synthesis
16
2
8
15
3
9
15
3
9
8
9
8
3
15
Martin, S.F. et al. Tetrahedron, 1999, 55, 3561.
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Macbecin 1 Evans Synthesis
4
12
4
13
5
5
12
13
Evans, D.A. et al. J. Org. Chem. 1993, 57, 1067.
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Macbecin 1 Panek Synthesis
14
10
5
14
5
10
10
14
5
Panek, J.S Xu, F. J. Am. Chem. Soc. 1995, 117,
19587.
21
Geldanamycin Andrus Synthesis
2
14
2
14
Andrus M.B. et al. Org. Lett. 2002, 4, 3549.
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Geldanamycin Synthesis
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Geldanamycin Synthesis
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Geldanamycin Synthesis
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Synthesis of Pyrone
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Geldanamycin Synthesis
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Geldanamycin Synthesis
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Geldanamycin Synthesis
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Geldanamycin Synthesis
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Geldanamycin Synthesis
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Geldanamycin Synthesis
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Geldanamycin Synthesis
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Geldanamycin Synthesis
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Geldanamycin Synthesis
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Evaluation of Geldanamycin as a Possible
Anti-Cancer Drug
Tyrosine kinase
Cancer cell proliferation
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Geldanamycin Binds a Specific Protein
Agarose Bead Geldanamycin
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994,
91, 8324.
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Cell Lysis to Obtain Cellular Contents
Cell lysis
Lysate obtained for study
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994,
91, 8324.
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Geldanamycin Binds a Specific Protein
Agarose Bead Geldanamycin
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994,
91, 8324.
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Cells Incubated with Geldanamycin
Cell lysis
Cells exposed to geldanamycin
Lysate obtained for study
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994,
91, 8324.
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Geldamycin Binding Occurs in vivo
Agarose Bead Geldanamycin
Lysate incubated with GA-coupled beads
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994,
91, 8324.
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Immunoblotting
Hsp90 monoclonal antibody added
Protein eluted from GA-coupled beads
Hsp90
control
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994,
91, 8324.
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Metabolic Labelling of Protein
Cells fed methionine containing
35S Protein labelled with 35S
Cell lysis
Lysate obtained for study
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994,
91, 8324.
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Radiolabelling
Cell lysate was incubated with GA-coupled beads
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994,
91, 8324.
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Radiolabelling
SDS-PAGE
1 2 3
1 2 Major cellular protein 3 After heat shock
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994,
91, 8324.
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Proteolytic Digestion
SDS-PAGE
V8

Proteins were partially digested with V8
proteolytic enzyme
1 2 3
RESULT all bands represent the same protein
RESULT Geldnamycin binds heat shock protein 90
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994,
91, 8324.
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Preclinical Studies of Geldanamycin
Anti-Tumor Activity Hepatotoxicity
Supko, J.G. et al. Cancer Chemo. Pharmacol. 1995,
36, 305.
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Structure Activity Relationship
19-substituents not tolerated
Schnur, R.C. et al. J. Med. Chem. 1995, 38, 3806.
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Structure Activity Relationship
Bicyclic and tricyclic quinones were active
19-substituents not tolerated
19
Schnur, R.C. et al. J. Med. Chem.1995, 38, 3806.
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Structure Activity Relationship
Bicyclic and tricyclic quinones were active
19-substituents not tolerated
17
19

• Small alkylamino groups
• unfunctionalized
• bearing hydroxy or
• amino groups

Schnur, R.C. et al. J. Med. Chem.1995, 38, 3806.
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Structure Activity Relationship
17
2
Carbamate and 2,3-double bond essential for
activity
3
17AAG 17-(allylamino)-17-demethoxygeldanamycin
Schnur, R.C. et al. J. Med. Chem. 1995, 38, 3813.
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Clinical Trials of 17AAG

• Prolonged disease stabilization
• Cytostatic drug effect

PHASE 1
Dose recommendations were made
PHASE 2

• 17AAG potential limitations
• Limited solubility
• Cumbersome formulation
• Dose and schedule dependant liver toxicity

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Genetic Plasticity of Cancer Cells
Steroid receptors
PR, AR, GCR
V-Src Akt Raf-1 Bcr-Abl
Mutant p53 HIF-1a
Hsp90
Soluble kinases
Transcription factors
ErbB2
Transmembrane kinases
Neckers, N. Trends Mol. Med. 2002, 8, S55.
53
Hsp90 Safeguards Against Mutations
Normal Protein Synthesis
Faulty Protein Synthesis
mutant p53
Benign mutation Normal Protein
Function Lethal mutation Cell Death
Neckers, L. et al. Proc. Natl. Acad. Sci. 1996,
93, 8379.
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Specificity of Hsp90 Inhibitors
Hsp90 is abundantly expressed in both normal and
tumor cells.
constitutively expressed
normal cell tumor cell
17AAG has up to 100x higher affinity for tumor
cells as compared to normal cells.
WHY?
normal cell tumor cell
Kamal, A. et al. Nature 2003, 425, 407.
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Future Work
Radiation therapy Chemotherapy Immunotherapy
Combination Therapy

17AAG
Improved pharmacological and toxicity profiles
Novel Hsp90 Inhibitors
Conformation of Hsp90-bound geldanamycin
Llauger, L. et al. J. Med. Chem. 2005, 48,
2892. Whitesell, L. Lindquist, S.L. Nature Rev.
Cancer 2005, 5, 761. Roe, S.M. et al. J. Med.
Chem. 1999, 42, 260.
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Summary

• Hsp90 inhibitors are a novel class of
  anti-cancer drugs.
• Geldanamycin has important functions as a
  chemical probe.
• SAR studies led to 17AAG phase 2 clinical
  trials.
• Novel Hsp90 inhibitors are currently being
  developed.

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Acknowledgements
Dr. Robert Ben Vincent Bouvet Frank Cease Jenn
Chaytor Pawel Czechura Jessica Jackman Nicole Le
Grand Aleks Paliga Suhuai Liu Roger Tam Indira
Thapa