Transcript Slide 1

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30/10/2009
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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
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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.”
NCI Website - http://www.cancer.gov/cancertopics/what-is-cancer
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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’.
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)
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Timeline of Cancer
 1761: Giovanni Morgagni of Padua was 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)
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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)
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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.
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.
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Targeting Cancer
All cancers share a common feature – rapid and
uncontrolled cell proliferation.
Normal Cell Cycle
Cancerous Cell Cycle
M
M
G0
G0
G2
G2
G1
G1
S
Cdc25
Phosphatase
S
Activates
Cdk
Cyc
Cell Cycle Regulator
CANCER
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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).
Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.
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Cdc25 Isoforms
In mammalian cells,three Isoforms have been identified :
Cdc25A, Cdc25B, Cdc25C
Boutros, R. et al. Nature Reviews Cancer, 2007, 7, 495-507.
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Activation of the Cdk/cyclin complex
Cell cycle progression requires activation of the
cyclin-dependent kinases(Cdk).
Myt1/Wee1
Cdk
Cyclin-Dependent Kinase
Cyc
Cyclin
CAK
Cdk Activating Kinase
CAK
Phosphorylation
Cdk
T161
Cdk
Cyc
T14 Y15
Dephosphorylation
T161
p p p
Cdk
Cyc
(Inactive)
Cyc
CDC25
p
Cdk
Cyc
p p
(Active)
Ducommun, B. et al. Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 818-824.
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Regulation of Cell Cycle Transition
 Different isoforms activate different complexes Cdc25B
Cdc25C
• Cdc25B
activates
Cdk1-CyclinB
at the centrosome
during the G2/M
transition.
M
Cdk1
CycB
Cdk1
CycB
G2
• Cdc25C activates
The Cdk1-CyclinB
complex in the nucleus
at the onset of mitosis.
• Cdc25A mainly controls
the G1/S Transitions via
the dephosphorylation
and activation of the
G1
Cdk2/CyclinE and
Cdk2/CyclinA
Cdk2
complexes.
CycE
Cdk2
CycA
S
Boutros, R. et al. Nature Reviews Cancer, 2007, 7, 495-507.
Cdc25A
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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
Ducommun, B. et al. Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 818-824.
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The Checkpoint Response
DNA Damage
DNA Damage
DNA Damage
Degradation via
proteosome
Cytoplasmic
Sequestration
Cytoplasmic
Sequestration
Ducommun, B. et al. Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 818-824.
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The Checkpoint Response
 Cell cycle arrest Cdc25B
Cdc25C
M
Cdk1
CycB
G1
Cdk1
Cdk2
CycB
CycE
G2
Cdk2
CycA
S
Cdc25A
Ducommun, B. et al. Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 818-824.
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Cdc25 overexpression causes Tumors
 Over-activation of Cdk-cyclin complexes – pushes cell cycle
in untimely manner.
Cdc25B
M
Cdc25C
Cdk1
CycB
Cdc25A overexpression
accelerates entry
into S-phase
G
1
Cdk1
Cdk2
CycB
CycE
Cdc25B overG2
expression rapidly
pushes the S or G2
phase cells into mitosis
even with incompletely
replicated DNA.
Cdk2
CycA
Boutros, R. et al. Nature Reviews Cancer, 2007, 7, 495-507.
S
Cdc25A
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Cdc25 overexpression: A recurring theme in
Cancer
Boutros, R. et al. Nature Reviews Cancer, 2007, 7, 495-507.
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Structure
N-terminal region
Regulatory Domain
C-terminal region
Catalytic Domain
 N-terminal regions are highly  C-terminal regions are highly
divergent
homologous
(~60% pairwise identity over
 Contains sites for
~200 amino acids)
phosphorylation
ubiquitination
 Contains the Catalytic Site
which regulate phosphatase
activity.
 The HCX5R motif
His – Cys – XXXXX – Arg
 Contains signals to control
conserved within the PTP
the intracellular localization
family
Rudolph, J. Biochemistry, 2007, 46, 3595-3604.
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Structure
Cdc25A
Cdc25B
(PDB ID: 1c25)
(PDB ID: 1qb0)
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Crystal Structure of Catalytic Domain of Cdc25B
Red – Active site
loop (HCX5R)
Blue - Sulfate
Side-view
Top-view
(PDB ID : 1qb0)
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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
Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.
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Crystal Structure of Catalytic Domain of Cdc25B
 HCX5R motif
 Histidine 472
 Cysteine 473
 Glutamic acid 474
 Phenylalanine 475
 Serine 476
 Serine 477
 Glutamic acid 478
 Arginine 479
H
C
X
R
 Backbone amides of five X
resides along with arginine
form multiple H-bonds with
the bound sulfate.
Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.
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Crystal Structure of Catalytic Domain of Cdc25B
 HCX5R motif
 Histidine 472
 Cysteine 473
 Glutamic acid 474
 Phenylalanine 475
 Serine 476
 Serine 477
 Glutamic acid 478
 Arginine 479
H
C
X
R
 The thiolate anion of
cysteine lies directly below
the bound sulfate.
Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.
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Crystal Structure of Catalytic Domain of Cdc25B
 The active site pocket is
small and extremely shallow.
Active
site
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.
Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.
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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
Rudolph, J. Mol Pharmacol. 2004, 66, 780-782.
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Crystal Structure of Catalytic Domain of Cdc25B
Catalytic pocket
Swimming pool
Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.
28
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Catalytic Mechanism
 Reaction mechanism for PTPs -
Chen, W. et al. Biochemistry, 2000, 39, 10781-10789.
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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.
Chen, W. et al. Biochemistry, 2000, 39, 10781-10789.
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Catalytic Mechanism
 Enzyme uses a monoprotonated substrate
The protein might use as its substrate a monoprotonated
phosphate in contrast to the typical bisanionic phosphate,
because of higher intrinsic reactivity.
Rudolph, J. et al. Biochemistry, 2002, 41, 14613-14623.
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Catalytic Mechanism
 Enzyme uses a monoprotonated substrate
Rudolph, J. et al. Biochemistry, 2002, 41, 14613-14623.
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Potential Druggable Targets for Cdc25
Enzyme
Activity
Transcription
Translation
Post – Translation
Protein-Protein
Interaction
Cdc25
Degradation
Subcellular
Localization
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
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Inhibitors of Cdc25
 Natural products
 Lipophilic acids
 Quinones
 Electrophiles
 Sulfonylated aminothiazoles
 Phosphate mimics
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
36
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Inhibitors of Cdc25
 Natural products
 Lipophilic acids
 Quinones
 Electrophiles
 Sulfonylated aminothiazoles
 Phosphate mimics
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
36
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Inhibitors of Cdc25
 Natural products
 Lipophilic acids
 Quinones
 Electrophiles
 Sulfonylated aminothiazoles
 Phosphate mimics
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
36
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Inhibitors of Cdc25
 Natural products
 Lipophilic acids
 Quinones
 Electrophiles
 Sulfonylated aminothiazoles
 Phosphate mimics
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
36
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Inhibitors of Cdc25
 Natural products
 Lipophilic acids
 Quinoids
 Electrophiles
 Sulfonylated aminothiazoles
 Phosphate mimics
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
36
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Inhibitors of Cdc25
 Natural products
 Lipophilic acids
 Quinoids
 Electrophiles
 Sulfonylated aminothiazoles
 Phosphate mimics
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
36
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Inhibitors of Cdc25
 Natural products
 Lipophilic acids
 Quinoids
 Electrophiles
 Sulfonylated aminothiazoles
 Phosphate mimics
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
36
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Inhibitors of Cdc25
 Natural products
 Lipophilic acids
 Quinones as Inhibitors of Cdc25B
 Electrophiles
 Sulfonylated aminothiazoles
 Phosphate mimics
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
36
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Quinones as Inhibitors of Cdc25B
Electrophilic properties of quinones suggest two possbile
interactions with enzyme :
 a sulfhydryl arylation of cysteine
 an ether linkage of serine
Can also oxidize the catalytic thiolate group of Cys473
Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
37
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Quinones as Inhibitors of Cdc25B
Naphthoquinones
Quinolinediones
Benzothiazole/
Benzoxazole –
diones
Indolyldihydroxyquinone
Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
38
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Quinones as Inhibitors of Cdc25B
 Naphthoquinones
IC50 = 3.8 μM*
Covalently inhibits enzyme
by arylating the catalytic
cysteine
NSC672121
NSC95397
IC50 = 0.125 μM*
* in-vitro IC50 values
Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
39
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Quinones as Inhibitors of Cdc25B
 Naphthoquinones
IC50 = 3.8 μM*
IC50 = 4.13 μM*
IC50 = 0.125 μM*
IC50 = 1.75 μM*
* in-vitro IC50 values
Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
40
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Quinones as Inhibitors of Cdc25B
 Naphthoquinones
IC50 = 12.9 μM*
IC50 = 4.1 μM*
IC50 = 10.3 μM*
IC50 = 1.8 μM*
* Growth inhibitory IC50 values for MCF7 human breast cancer
cell lines
Peyregne, V. P. et al. Mol. Cacncer Ther., 2005, 4, 595-602.
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Quinones as Inhibitors of Cdc25B
 Naphthoquinones
Hydrogen bonding between the enolic anion
and the hydroxy group
Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
42
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Quinones as Inhibitors of Cdc25B
 Naphthoquinones
Binding Mode
NSC 128981
IC50 = 0.62 μM
Result of 50 independent Autodock and GOLD docking runs –
ΔGbind
Ligand
Ntot
focc
(kcal/mol)
NSC 128981
11
11
-7.89
Lavechhia, A. et al. Chem Med Chem, 2006,1, 540-550.
GOLD score
52.28
43
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Quinones as Inhibitors of Cdc25B
 Quinolinediones
IC50 = 0.21 μM
NSC 663284
Inhibits enzyme in both reversible and irreversible manner.
* in-vitro IC50 value
Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
44
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Quinones as Inhibitors of Cdc25B
 Quinolinediones
Chlorine moiety
is not required
IC50 = 0.21 μM
* in-vitro IC50 values
Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
45
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Quinones as Inhibitors of Cdc25B
 Quinolinediones
Chlorine moiety
is not required
IC50 = 0.21 μM
2-morpholin-4-ylethylamino
moiety increases activity
Decreased activity when  substituted with different groups
 shifted to 6-position (IC50 = 20μM)
* in-vitro IC50 values
Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
45
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Quinones as Inhibitors of Cdc25B
 Quinolinediones
R = 2-Me : IC50 = 4.6 μM
R = 4-Me : IC50 = 4.6 μM
Chlorine moiety
is not required
R = 2-CN : IC50 = 3.7 μM
Small groups
are tolerated
IC50 = 0.21 μM
Aza analogues are less active
2-morpholin-4-ylethylamino
moiety increases activity
* in-vitro IC50 values
Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
45
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Quinones as Inhibitors of Cdc25B
 Quinolinediones
Binding Mode
Two modes were observed –
 Autodock placed the quinolinequinone
ring into the “swimming pool” cavity
 GOLD placed quinolinequinone ring
into the catalytic site
NSC 663284
IC50 = 0.21 μM
Result of 50 independent Autodock and GOLD docking runs –
ΔGbind
Ligand
Ntot
focc
(kcal/mol)
NSC 663284
10
26
-8.12
Lavechhia, A. et al. Chem Med Chem, 2006,1, 540-550.
GOLD score
42.97
46
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Quinones as Inhibitors of Cdc25B
 Quinolinediones
Binding Mode
Autodock
Lavechhia, A. et al. Chem Med Chem, 2006,1, 540-550.
GOLD
47
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Quinones as Inhibitors of Cdc25B
 Indolyldihydroxyquinones
 Mode of action different from other quinones – Reversible
and non-covalent inhibitors.
 Two electron donating hydroxy groups and elctron donating
indole substituent, making them much less likely to accept
nucleophiles.
Sohn, J. et al. J. Med. Chem., 2003, 46, 2580-2588.
48
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Quinones as Inhibitors of Cdc25B
 Indolyldihydroxyquinones
Substitution
reduced activity
 Halides and benzyloxy
increase potency
 Methyl is deleterious
IC50 = 18 μM
5
4
Substituents of
size greater than
propyl increase
potency
6
7
2
Methyl group is
tolerated
Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
Substitution
reduced activity
49
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Quinones as Inhibitors of Cdc25B
 Indolyldihydroxyquinones
Binding Mode
Two modes were observed –
 Autodock placed the quinone ring into
the “swimming pool” cavity
 GOLD placed quinone ring into the
catalytic site
Compound 1
IC50 = 1 μM
Result of 50 independent Autodock and GOLD docking runs –
ΔGbind
Ligand
Ntot
focc
(kcal/mol)
Compound 1
11
11
-7.89
Lavechhia, A. et al. Chem Med Chem, 2006,1, 540-550.
GOLD score
52.28
50
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Quinones as Inhibitors of Cdc25B
 Indolyldihydroxyquinones
Binding Mode
Autodock
Lavechhia, A. et al. Chem Med Chem, 2006,1, 540-550.
GOLD
51
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Quinones as Inhibitors of Cdc25B
 Benzothiazole- and Benzoxazole- diones
IC50 = 0.25 μM
 Irreversible inhibition
IC50 = 0.15 to 0.44 μM
Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
52
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Pharmacophoric Model for Cdc25B Reversible
Inhibition
Acceptor Hbond group
A B
D
C
Acceptor Hbond group
Catalytic pocket
Swimming pool
Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.
53
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Pharmacophoric Model
Acceptor Hbond group
A B
D
C
Acceptor Hbond group
Catalytic pocket
Swimming pool
Group B : Core structure, mostly quinone
Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.
53
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Pharmacophoric Model
Acceptor Hbond group
A B
D
C
Acceptor Hbond group
Catalytic pocket
Swimming pool
Group A : A bulky aromatic system
Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.
53
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Pharmacophoric Model
Acceptor Hbond group
A B
D
C
Acceptor Hbond group
Catalytic pocket
Swimming pool
Group C : An aromatic ring or acceptor H-bond group
Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.
53
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Pharmacophoric Model
Acceptor Hbond group
A B
D
C
Acceptor Hbond group
Catalytic pocket
Swimming pool
Linker : An alkylic chain of 3-4 units
Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.
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Future Prospects
Substrate Recognition Site -
Lack of any apparent substrate
recognition site in the catalytic
loop.
The C473S mutant binds tightly
to Cdk2-pTpY – CycA.
Three hotspot residues located
>20Å from the active site, mediate
protein substrate recognition.
Sohn, J. et al. PNAS, 2004, 101, 16437-16441.
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Future Prospects
Substrate Recognition Site  R488L and Y497A mutants
reduced the kcat/Km for Cdk2-pTpY
– CycA, while retaining the activity
towards the small-molecule
substrates.
 R492L mutation showed similar
results.
Arg 488
Arg 492
Tyr 497
Sohn, J. et al. PNAS, 2004, 101, 16437-16441.
Rudolph, J. Biochemistry, 2007, 46, 3595-3604.
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Future Prospects
Substrate Recognition Site –
Docking model of Cdc25B
with its protein substrate
Cdk2-pTpY–CycA showed
the three hotspot residues –
Arg488, Arg492 and Tyr497
interacting with the two aspartate
residues of Cdk2.
Rudolph, J. Biochemistry, 2007, 46, 3595-3604.
Cdc25B: magenta
Cdk2-pYpY–CycA : blue
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Future Prospects
 A potential binding pocket
Binding of suitable ligands
could engage the substrates
involved in substrate recognition
and interfere in enzyme/substrate
association.
Arg 488
Arg 492
Tyr 497
Rudolph, J. Biochemistry, 2007, 46, 3595-3604.
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Future Prospects
 Peptide Derived Inhibitors
Inhibitors designed based on sequence homology with
the protein substrate.
Active site peptide ligand
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
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Future Prospects
 Is activating Cdc25 Phosphatase a feasible approach?
Moderate increase in levels of Cdc25B have shown to
significantly increase the sensitivity of tumor cells to
doxorubicin or ionizing radiations.
 Idea would be to radiosensitize or chemosensitize
cancer cells and push them to commit suicide.
 High risk factor to patient.
Boutros, R. et al. Nature Reviews Cancer, 2007, 7, 495-507.
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 Cdc25 Phosphatases represent a good target for
developing novel anticancer drugs.
 Scope for developing novel strategies to target them.
 Crystal structures of Cdc25A and Cdc25B provide a
rational basis for the design of potent and selective
inhibitors.
 Further improvement of these inhibitory compounds is
likely to lead to their introduction in human clinical trials.
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 Dr. Glen Kellogg
 Kellogg’s Molecular Modeling & Drug Design Group
 Department of Medicinal Chemistry
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