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Geldanamycin: A Novel Approach to Treating Cancer
Elisabeth von Moos
December 1, 2005
2
• More than 100 different and distinct diseases.
What is Cancer?
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
Chemotherapy
Current Cancer Therapies
Surgery
Radiation therapy
Biological therapy
Photodynamic therapy
Targeted cancer therapy
Many cancers have no cure.
Poor prognosis for patients with solid tumors.
4
Important Therapeutic Targets
Therapy Mutant p53
HIF-1
ErbB2
V-SrcAktRaf-1Bcr-Abl
Solublekinases
Transmembrane kinases
Transcription factors
Neckers, N. Trends Mol. Med. 2002, 8, S55.
Steroid receptors
PR, AR, GCR
5
Heat Shock Protein 90
Hsp90 Mutant p53
HIF-1
ErbB2
V-SrcAktRaf-1Bcr-Abl
Solublekinases
Transmembrane kinases
Transcription factors
Neckers, N. Trends Mol. Med. 2002, 8, S55.
Steroid receptors
PR, AR, GCR
6
Heat Shock Proteins
Prevent protein aggregation
Intracellular proteintransport
Fold nascentproteins or repair misfolded proteins
Repair denaturedproteins
Control regulatoryproteins
Degrade denatured proteins
7
Central Dogma of Molecular Biology
DNA
RNA
Protein
Transcription
Translation
8
Molecular Chaperones
+ + nascent protein mature protein
ATP ADP + Pi
9
Heat Shock Proteins Protect Cells
stress
Denatured proteins detected
Heat shock proteins produced
Denatured proteins refolded
Stress denatures proteins
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
Prodromou, C. Nature Struct. Biol. 1997, 4, 477.
ATP
11
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
Heat Shock Protein 90
Prodromou, C. Nature Struct. Biol. 1997, 4, 477.
ADP
12
Mechanism of Action of Hsp90
Isaacs, J.S. Cancer Cell, 2003, 3, 213. Proteosome
client
client
Hip
Hsp70
Hsp40
p60Hop
BAG-1
PP5
ImmunoP’s
p23
Cyp40
p50Cdc37
ADPATP
ATP hydrolysis
ATP exchange
unfolded
folded
13
The Ansamycin Antibiotics
O
ONH
O
O
MeO
MeOR2
Me
MeO
Me
MeR3
NH2
O
R1
14
Geldanamycin
DeBoer, C. et al. J. Antibiot. 1970, 33, 781. Merrell, P.H. et al. J. Am. Chem. Soc. 1970, 92, 7591.
O
ONH
O
O
MeOOMe Me
NH2
O
MeO
O
ONH
O
O
MeOOH
MeO
MeO
NH2
O
H
O
ONH
O
O
MeOOMeMeO
NH2
O
MeO
• Isolated in 1970 fromStreptomyces hygroscopicus• Shows anti-microbial, anti-viral, and anti-tumor activity
15
Macbecin 1
O
ONH
O
O
MeOOMe Me
NH2
O
MeO
O
ONH
O
O
MeOOH
MeO
MeO
NH2
O
O
ONH
O
O
MeOOMeMeO
NH2
O
MeO
Tanida, S. et al. J. Antibiot. 1980, 33, 199. Muroi, M. et al. Tetrahedron, 1981, 37, 1123.
• Isolated in 1980 from Nocardia• Shows anti-microbial, anti-viral, and anti-tumor activity
16
Herbimycin A
O
ONH
O
O
MeOOMe Me
NH2
O
MeO
O
ONH
O
O
MeOOH
MeO
MeO
NH2
O
O
ONH
O
O
MeOOMeMeO
NH2
O
MeO
Tanida, S. et al. J. Antibiot. 1980, 33, 199. Muroi, M. et al.Tetrahedron, 1981, 37, 1123.
• Isolated in 1980 from Nocardia• Shows anti-microbial, anti-viral, and anti-tumor activity
17
Herbimycin A – Tatsuta Synthesis
Tatsuta, K. et al. Tetrahedron Lett. 1991, 32, 6015.
O
ONH
O
O
MeOOMeMeO
NH2
O
MeO
OHC CHOOMe
OMe
OMe
LiOMe
NHTr
OHCOMe
OMe
OHCOMe OTBS
OMe
O
OMe
OH
HOHO
HO
+
15
16
34
154
18
Herbimycin A – Martin Synthesis
O
ONH
O
O
MeOOMeMeO
NH2
O
MeO OMe
LiOMe
NR2O O
OMe
MeOH
OTBDMS
OMe
+
OCHO
MeOH
OTBDMS
LiO
OMe
OMe
+
Martin, S.F. et al. Tetrahedron, 1999, 55, 3561.
OXn
OOHO
OR
OH
+
16
15
2
3
15
89
8
93
3159 8
19
Macbecin 1 – Evans Synthesis
O
O
MeOOMeMeO
NH2
O
MeO
O
ONH
MeOP(OCH2CF3)2
OO
HN
OMe
MeO
OTBSOOMe
N S
O S
OMe
OMe
OMeO2N
Evans, D.A. et al. J. Org. Chem. 1993, 57, 1067.
12
134
5
12
13
5
4
20
Macbecin 1 – Panek Synthesis
O
ONH
O
O
MeOOMe
NH2
O
MeO
OMeO2N
OMe
OMe
OMe
CO2MeMe2SiPh
Bn
OMeO2N
OMe CO2Et
OMe OMe OPMB
OMe
Me2SiPh
OMe
CO2MeMe2SiPh
Panek, J.S; Xu, F. J. Am. Chem. Soc. 1995, 117, 19587.
14
14
10
10
5
5
14
10 5
21
Geldanamycin – Andrus Synthesis
Andrus M.B. et al. Org. Lett. 2002, 4, 3549.
O
ONH
O
O
MeOOH
MeO
MeO
NH2
O
ArOMe
OTBSCHO
Ar CHO
CO2H
OMe
OTBS OTES
OMe
OMe
ONH2
MeO
Me
OO
O
RR
+
2
2
14
14
22
Geldanamycin Synthesis
OMeMeO
OMe
OMe
NO2
MeO CHO
OMe
1) BuLi, DMF 68%2) HNO3 AcOH 83%
MeO
NO2
MeO
OMe
Br
1) NaBH4 98%2) PBr3, pyridine 93%
N O
OO
Bn
MeO
NO2
MeO
OMe
N O
OO
Bn
NHMDS, THF
-78oC 88% >19:1
23
Geldanamycin Synthesis
N O
OO
Bn
MeO
NO2
MeO
OMe
CNHO
MeO
NO2
MeO
O
CN
Me
1) LiBH4 96%2) DEAD, PPh3
81%
DIBALH2O 92%
MeO
NO2
MeO
O
CHO
Me
24
Geldanamycin Synthesis
MeO
NO2
MeO
OOH
OO
O
PhOMePhOMe
Me
MeO
NO2
MeO
O
CHO
Me
OO
O
PhOMePhOMe
+
(c-Hex)2BOTf
Et3N, CH2Cl2
-78oC
70% 10:1
25
Synthesis of Pyrone
MeO
OMe
Ot-BuBr
OO
O
O
PhOMePhOMe
MeO
OMeOH
OH
AD-mix-t-BuOH:
H2O 1:1
81% >95%ee
n-Bu2SnO
PhH,
Bu4N+I-, TFA 80%
26
MeO
NO2
MeO
OOH
OO
O
PhOMePhOMe
Me
MeO
NO2
MeO
O
CHO
Me
OO
O
PhOMePhOMe
+
(c-Hex)2BOTf
Et3N, CH2Cl2
-78oC
70% 10:1
Geldanamycin Synthesis
MeO
NO2
MeO
OOMe
CO2Me
O OH
MeOPh PhOMe
1) Me3OBF4 91%2) NaOMe, MeOH 90%
Me
27
Geldanamycin Synthesis
OMe
OTBS
ArOH
1) DIBAL 90%2) AlMe3 80%
MeO
NO2
MeO
OOMe
CO2Me
O OH
MeOPh PhOMe
MeOMe
CO2Me
OTBS
Ar
1) CAN 93%2) TBSCl, imid. 93%
OMe
OTBS
Ar
1) DMP 93%2) Ph3P=CH2 95%
28
Geldanamycin Synthesis
OMe
OTBS
ArCO2Et
Ph3P CO2Et
1) TBSCl, imid. 2) CSA 76% (2 steps)3) DMP 94%4)
98% 16:1
OMe
OH
Ar
OH
OMe
OTBS
Ar
1) HF.pyr 97%
2) BH3.THF
95% 5:1
OMe
OTBS
ArCHO
1) DIBAL2) Swern 77% (2 steps)
29
OMe
OTBS
ArCO2Me
OMe
OH1) LiOH2) TMSCHN2
PhH:MeOH 4:187% (2 steps)
Geldanamycin Synthesis
OMe
OTBS
ArCHO
OMeO
O
NSO2MesBn
MePh
c-Hex2BOTf, Et3N
-78oC 90% > 20:1OMe
OTBS
Ar Aux
O
OMe
OH
30
Geldanamycin Synthesis
OMe
OTBS
Ar
OTES
OMe CO2Me
OMe
OTBS
Ar
OHCO2Me
OMe
P CO2Me(F3CCH2O)2
O
1) TESOTf, lut. 95%2) DIBAL3) KH, 18-crown-6
13:1 81%
31
Geldanamycin Synthesis
OMe
OTBS
Ar
OTES
OMe CO2Me
OMe
OTBS
Ar
OTES
OMe
O O-allyl
(EtO)2P
O
O O
1) DIBAL 91%2) DMP 89%3)
DBU, LiCl 19:1 93%
32
Geldanamycin Synthesis
OMe
OTBS OTES
OMe
O O-allyl
MeO
NO2
MeO
OMe
OMe
OTBS OTES
OMe
O OH
MeO
NH2
MeO
OMe
1) NaBH4.S
2) Pd(PPh3)4
morph. 85%
33
Geldanamycin Synthesis
OMe
OTBS OTES
OMe
CO2H
OMe
NH2
MeO
OMe
OMe
OMeNH
O
OTES
MeOOTBS
MeO
MeO
OMe
OMeNH
O
O
MeOOH
MeO
MeO
NH2
O
BOP-Cl, i-Pr2NEt
tol. 85oC 76%
1) TBAF 90%2) Cl3CCONCO/MeOH K2CO3 89%3) HF 95%
34
Geldanamycin SynthesisOMe
OMeNH
O
O
MeOOH
MeO
MeO
NH2
O
O
ONH
O
O
MeOOH
MeO
MeO
NH2
O
O
OMeNH
O
O
MeOOH
O
MeO
NH2
O
+
1:10
HNO3AcOH55%
35
Evaluation of Geldanamycin as a Possible Anti-Cancer Drug
O
ONH
O
O
MeOOH
MeO
MeO
NH2
O
Cancer cellproliferation
Tyrosine kinase
36
Geldanamycin Binds a Specific Protein
GA
GA
GA
GA
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994, 91, 8324.
Agarose BeadGeldanamycinGA
37
Cell Lysis to Obtain Cellular Contents
Cell lysis
Lysate obtained for study
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994, 91, 8324.
38
Geldanamycin Binds a Specific Protein
Agarose BeadGeldanamycin
GA
GA
GA
GA
GA
GA
GA
GA
GA
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994, 91, 8324.
39
Cells Incubated with Geldanamycin
Cell lysis
GA
GA
GAGA
Cells exposed togeldanamycin
Lysate obtained for study
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994, 91, 8324.
GA
GA
GAGA
40
Geldamycin Binding Occurs in vivo
Agarose BeadGeldanamycin
GA
GA
GA
GA
GA
Lysate incubated with GA-coupled beads
GA
GA
GA
GA
GA
GA
GA
GA
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994, 91, 8324.
41
Immunoblotting
GA
GA
GA
GA
control
Hsp90 monoclonal antibody added
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994, 91, 8324.
Hsp90Protein eluted fromGA-coupled beads
42
Metabolic Labelling of Protein
35S
35S
35S
35S
35S 35S
35S35S
35S
Cell lysis
Cells “fed” methioninecontaining 35S
Protein labelled with 35S Lysate obtained for study
35S
35S
35S
35S
35S 35S
35S35S
35S
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994, 91, 8324.
43
Radiolabelling
GA
GA
GA
GA
35S
35S
35S
35S
GA
GA
GA
GA
Cell lysate was incubated with GA-coupled beads
35S
35S
35S
35S35S
35S35S
35S35S
35S
35S35S 35S
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994, 91, 8324.
44
12 Major cellular protein3 After heat shock
Radiolabelling
1 2 3
GA
GA
GA
GA
35S
35S
35S
35S
35S
35S
35S
35S
35S
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994, 91, 8324.
SDS-PAGE
45
Proteolytic Digestion
SDS-PAGE
RESULT: all bands represent the same protein
V8
Proteins were partially digested with V8 proteolytic enzyme
=
Whitesell, L. et al. Proc. Natl. Acad. Sci. 1994, 91, 8324.
RESULT: Geldnamycin binds heat shock protein 90
1 2 3
46
Preclinical Studies of Geldanamycin
Anti-Tumor Activity
Hepatotoxicity
O
ONH
O
O
MeOOH
MeO
MeO
NH2
O
Supko, J.G. et al. Cancer Chemo. Pharmacol. 1995, 36, 305.
47
O
ONH
O
O
MeOOH
MeO
MeO
NH2
O
Structure Activity Relationship
19-substituents not tolerated
Schnur, R.C. et al. J. Med. Chem. 1995, 38, 3806.
48
Structure Activity Relationship
Bicyclic and tricyclic quinoneswere active
19-substituents not tolerated
Schnur, R.C. et al. J. Med. Chem.1995, 38, 3806.
19
ONH
O
O
MeOOH MeO
NH2
O
NN
NMe
Me
49
O
ONH
O
O
MeOOH
HN
MeO
NH2
O
Structure Activity Relationship
Small alkylamino groups:- unfunctionalized- bearing hydroxy oramino groups
Schnur, R.C. et al. J. Med. Chem.1995, 38, 3806.
19-substituents not tolerated
Bicyclic and tricyclic quinoneswere active
1917
50
O
ONH
O
O
MeOOH
HN
MeO
NH2
O
H
O
ONH
O
O
MeOOH
HN
MeO
NH2
O
H
Structure Activity Relationship
17AAG: 17-(allylamino)-17-demethoxygeldanamycin
Schnur, R.C. et al. J. Med. Chem. 1995, 38, 3813.
Carbamate and 2,3-double bond essential for activity
17
23
51
17AAG potential limitations:
• Limited solubility• Cumbersome formulation• Dose and schedule dependant liver toxicity
PHASE 2
Clinical Trials of 17AAG
Prolonged disease stabilization• Cytostatic drug effect
PHASE 1
Dose recommendations were made
52
Genetic Plasticity of Cancer Cells
Hsp90 Mutant p53
HIF-1
ErbB2
V-SrcAktRaf-1Bcr-Abl
Solublekinases
Transmembrane kinases
Transcription factors
Neckers, N. Trends Mol. Med. 2002, 8, S55.
Steroid receptors
PR, AR, GCR
53
Benign mutation Normal Protein FunctionLethal mutation Cell Death
Hsp90 Safeguards Against Mutations
Normal Protein Synthesis
Faulty Protein Synthesis
Neckers, L. et al. Proc. Natl. Acad. Sci. 1996, 93, 8379.
mutant p53
54
Specificity of Hsp90 Inhibitors
17AAG has up to 100x higher affinity for tumor cells as compared to normal cells.
Hsp90 is abundantly expressed in both normal and tumor cells.
normal cell tumor cell
constitutively expressed
WHY?
normal cell tumor cellKamal, A. et al. Nature 2003, 425, 407.
55
Future Work
Improved pharmacologicaland toxicity profiles
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.
17AAGRadiation therapyChemotherapyImmunotherapy
Novel Hsp90 Inhibitors
CombinationTherapy
+
Conformation of Hsp90-bound geldanamycin
56
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.
57
Acknowledgements
Dr. Robert BenVincent BouvetFrank CeaseJenn ChaytorPawel CzechuraJessica JackmanNicole Le GrandAleks PaligaSuhuai LiuRoger TamIndira Thapa
