Presented By : HARDIK PARIKH Department of Medicinal Chemistry
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Transcript of Presented By : HARDIK PARIKH Department of Medicinal Chemistry
Presented By:HARDIK PARIKH
Department of Medicinal ChemistryInstitute for Structural Biology and Drug Discovery
Virginia Commonwealth Universityemail: [email protected]
CDC25 PHOSPHATASE:A POTENTIAL TARGET FOR NOVEL
ANTICANCER AGENTS
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
2
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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 3
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Timeline of Cancer
3000BC: Earliest observations of cancerBone 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.
4
Origin of word CancerCredited 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.aspImages adapted from – http://www.cancerquest.org (accessed 10/22/09)
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ACS Website- http://www.cancer.org/docroot/CRI/content/CRI_2_6x_the_history_of_cancer_72.aspImages adapted from – http://www.cancerquest.org (accessed 10/22/09)
1914: Mutation theory of cancerTheodor Boveri proposed the Somatic
Mutation Theory of Cancer. He believed that cancer was caused by abnormal chromosomes.
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 TreatmentWilliam Halsted, the first professor of surgery at John Hopkins, Harvard, and Yale, performed the first radical mastectomy.
5
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ACS Website- http://www.cancer.org/docroot/CRI/content/CRI_2_6x_the_history_of_cancer_72.aspImages adapted from – http://www.cancerquest.org (accessed 10/22/09)
Timeline of Cancer
2003: Human Genome ProjectIdentified ~25,000 genes in human DNA.
1940s: Era of Cancer Chemotherapy Goodman and Gilman suggested that
nitrogen mustards could be used to treat lymphoma.
2006: First cancer vaccineFDA approved Gardasil, a vaccine that
protects against HPV – Human papillomavirus, major cause for cervical cancer.
6
1971: War on Cancer declared by President NixonThe National Cancer Act was signed into law; additional
$100 million funds released to find a cure for cancer.
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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. 7
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.
CANCER
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Targeting Cancer
G2
M
S
G0
G1
Normal Cell Cycle
All cancers share a common feature – rapid and uncontrolled cell proliferation.
G0
G1
G2
M
S
Cancerous Cell Cycle
CANCER
Misregulation/
Over activationCdc25Phosphatase Activates
8
CycCdk
Cell Cycle Regulator
Regulat
ed
activit
y
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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).
9Reynolds, 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
10Boutros, R. et al. Nature Reviews Cancer, 2007, 7, 495-507.
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Cell Cycle Regulation byCdc25 Phosphatases
11
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Cell cycle progression requires activation of the cyclin-dependent kinases(Cdk).
Activation of the Cdk/cyclin complex
p
Myt1/Wee1
T14
pY15
(Inactive)
12Ducommun, B. et al. Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 818-824.
CycCdk
Cyclin-Dependent Kinase
Cyc
Cdk
Cyclin
Cdk
Cyc
CAK Cdk Activating Kinase
p
CycCdk
T161
CAKPhosphorylation
(Active)
CDC25
p p
p
CycCdk
T161Dephosphorylation
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• Cdc25B activates Cdk1-CyclinB at the centrosomeduring the G2/Mtransition.
• Cdc25C activates The Cdk1-CyclinB complex in the nucleus at the onset of mitosis.
Cdc25B
Cdc25C
Cdc25A
• Cdc25A mainly controls the G1/S Transitions via the dephosphorylation
and activation of the Cdk2/CyclinE and
Cdk2/CyclinAcomplexes.
Regulation of Cell Cycle Transition
Different isoforms activate different complexes -
13Boutros, R. et al. Nature Reviews Cancer, 2007, 7, 495-507.
G1
G2
M
S
CycB
Cdk1
CycBCdk1
CycE
Cdk2
CycA
Cdk2
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The Checkpoint Response
Checkpoint Kinase 1
Checkpoint Kinase 2
Mitogen-activated Protein Kinase Activated Protein Kinase 2 /MAPKAP Kinase2
Cdc25 Phosphatases
14Ducommun, B. et al. Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 818-824.
DNA Damage
Degradation via proteosome
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The Checkpoint Response DNA Damage DNA Damage DNA Damage
Ducommun, B. et al. Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 818-824.
Degradation via proteosome
Cytoplasmic Sequestration
Cytoplasmic Sequestration
15
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Cdc25B
Cdc25C
Cdc25A
16
G1
G2
M
S
CycB
Cdk1
CycBCdk1
CycE
Cdk2
CycA
Cdk2
The Checkpoint Response
Cell cycle arrest -
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.
Cdc25A overexpression accelerates entry
into S-phase
Cdc25B over-expression rapidly pushes the S or G2phase cells into mitosiseven with incompletely replicated DNA.
17Boutros, R. et al. Nature Reviews Cancer, 2007, 7, 495-507.
Cdc25A
G1
G2
M
S
CycB
Cdk1
CycBCdk1
CycE
Cdk2
CycA
Cdk2
Cdc25B
Cdc25C
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Cdc25 overexpression: A recurring theme in Cancer
18Boutros, R. et al. Nature Reviews Cancer, 2007, 7, 495-507.
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Structure ofCdc25 Phosphatases
19
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Structure
C-terminal regionCatalytic Domain
N-terminal regionRegulatory Domain
N-terminal regions are highly divergent
Contains sites for phosphorylationubiquitinationwhich regulate phosphataseactivity.
Contains signals to control the intracellular localization
C-terminal regions are highly homologous
(~60% pairwise identity over ~200 amino acids)
Contains the Catalytic Site
The HCX5R motifHis – Cys – XXXXX – Arg
conserved within the PTP family
20Rudolph, J. Biochemistry, 2007, 46, 3595-3604.
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Structure
Cdc25A(PDB ID: 1c25)
Cdc25B(PDB ID: 1qb0)
21
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Crystal Structure of Catalytic Domain of Cdc25B
Side-view Top-view(PDB ID : 1qb0)
Red – Active siteloop (HCX5R)Blue - Sulfate
22
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Top-view
Red – Active siteloop (HCX5R)Blue - Sulfate
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.
23Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.
Crystal Structure of Catalytic Domain of Cdc25BFor more presentations and information visit http://www.pharmaxchange.info
HCX5R motif Histidine 472
H Cysteine 473 C Glutamic acid 474 Phenylalanine 475 Serine 476 X Serine 477 Glutamic acid 478 Arginine 479 R
Backbone amides of five X resides along with arginine form multiple H-bonds with the bound sulfate. 24Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.
Crystal Structure of Catalytic Domain of Cdc25BFor more presentations and information visit http://www.pharmaxchange.info
HCX5R motif Histidine 472
H Cysteine 473 C Glutamic acid 474 Phenylalanine 475 Serine 476 X Serine 477 Glutamic acid 478 Arginine 479 R
The thiolate anion of cysteine lies directly below the bound sulfate.
25Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.
Crystal Structure of Catalytic Domain of Cdc25BFor more presentations and information visit http://www.pharmaxchange.info
Active site The active site pocket is
small and extremely shallow.
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.
26Reynolds, R. A. et al. Mol. Biol., 1999, 293, 559-568.
Crystal Structure of Catalytic Domain of Cdc25BFor more presentations and information visit http://www.pharmaxchange.info
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 cysteineRed – Water molecules
SwimmingPool
27Rudolph, J. Mol Pharmacol. 2004, 66, 780-782.
Crystal Structure of Catalytic Domain of Cdc25BFor more presentations and information visit http://www.pharmaxchange.info
HS
Cys473
Phe475
Arg544
NH
H2N NH
OH
Tyr428
Phe543
Thr547 CH3
OH
NH
Trp550
Arg482
HN
NH2HN
Swimming poolCatalytic pocket
Arg479
NHH2N
NH
28Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.
Crystal Structure of Catalytic Domain of Cdc25BFor more presentations and information visit http://www.pharmaxchange.info
Catalytic Mechanism ofCdc25 Phosphatases
29
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Catalytic Mechanism
Reaction mechanism for PTPs -
CysS
NHN
NH2
H H
Arg
OOH
Acid
P
O
O O
OSubstrate
CysS
NHN
NH2
H H
Arg
OO
Acid
PO
O
O
O
Substrate
H
H2O
HO Substrate
CysS
NHN
NH2
H H
Arg
OO
Acid
P
O
O
O
O HHP
OHO
OO
30Chen, 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.
31Chen, 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.
P
O
O O
HOSubstrateP
O
O O
OSubstrate
32Rudolph, J. et al. Biochemistry, 2002, 41, 14613-14623.
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CysS
NHN
NH2
H H
Arg
P
O
O O
HOSubstrate
Catalytic Mechanism
Enzyme uses a monoprotonated substrate
CysS
NHN
NH2
H H
Arg
PO
O O
O
Substrate
H
CysS
NHN
NH2
H H
Arg
PO
O O
O
Substrate
H
HO Substrate
H2O
CysS
NHN
NH2
H H
Arg
P O
O
OO
H
H
POHO
O OH
33Rudolph, J. et al. Biochemistry, 2002, 41, 14613-14623.
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Small MoleculeInhibitors of
Cdc25 Phosphatases
34
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Translation
Post – Translation
Protein-Protein Interaction
Subcellular Localization
Degradation
Transcription
Potential Druggable Targets for Cdc25
Enzyme Activity
Cdc25
35Lazo, 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
36Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
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Inhibitors of Cdc25
N
N OO
OH2N
HO
NC
H
H
NMe
OH
O
O
OH
H
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 Cdc25OH
O
S
O
C9H19
O
NH
COOH
N
O
HN
ON
OPh
Ph
Ph
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
36
Natural products
Lipophilic acids
Quinones
Electrophiles
Sulfonylated aminothiazoles
Phosphate mimics
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Natural products
Lipophilic acids
Quinones
Electrophiles
Sulfonylated aminothiazoles
Phosphate mimics
Inhibitors of Cdc25
O
NNH
N
O
O
Cl
O
O
OH
HO
HN
Cl
Cl
H3C
O
O
SHO
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
36
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Inhibitors of Cdc25
N
O
O
S
S
HO
HO
NH
N
O
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
36
Natural products
Lipophilic acids
Quinoids
Electrophiles
Sulfonylated aminothiazoles
Phosphate mimics
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Inhibitors of Cdc25
S
NNH
S
OO
S
NNH
S
OO
Cl
Cl
Cl
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
36
Natural products
Lipophilic acids
Quinoids
Electrophiles
Sulfonylated aminothiazoles
Phosphate mimics
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Inhibitors of Cdc25
O P
O
OHOH
N
O
HN
O
O
O
HO
HO O
O
O
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
36
Natural products
Lipophilic acids
Quinoids
Electrophiles
Sulfonylated aminothiazoles
Phosphate mimics
For more presentations and information visit http://www.pharmaxchange.info
Inhibitors of Cdc25
O
NNH
N
O
O
Cl
O
O
OH
HO
HN
Cl
Cl
H3C
O
O
SHO
Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
36
Natural products
Lipophilic acids
Quinones as Inhibitors of Cdc25B
Electrophiles
Sulfonylated aminothiazoles
Phosphate mimics
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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
O
O
37Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
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Quinones as Inhibitors of Cdc25B
O
O
R
R
R
Naphthoquinones
N
O
O
R
R
R
Quinolinediones
O
O
OH
HO
HN
Indolyldihydroxy-quinone
X
N
O
O
R
R
RX = S,O
Benzothiazole/Benzoxazole –
diones
O
O
38Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
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Quinones as Inhibitors of Cdc25B
Naphthoquinones
O
O
S
CH3
OH
IC50 = 3.8 μM*
* in-vitro IC50 values
39
NSC672121O
O
S
S
OH
OH
IC50 = 0.125 μM*
NSC95397
Covalently inhibits enzyme by arylating the catalytic
cysteine
Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
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Quinones as Inhibitors of Cdc25B
Naphthoquinones
O
O
S
CH3
OH
IC50 = 3.8 μM*
O
O
S
S
OH
OH
IC50 = 0.125 μM*
O
O
S
CH3
OH
O
IC50 = 4.13 μM*
O
O
S
S
OH
OH
O
O
IC50 = 1.75 μM*
* in-vitro IC50 values
40Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
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Quinones as Inhibitors of Cdc25B
Naphthoquinones
O
O
S
CH3
OH
IC50 = 12.9 μM*
O
O
S
S
OH
OH
IC50 = 10.3 μM*
O
O
S
S
OH
OH
OH
O
O
S
S
OH
OH
OH
OH
IC50 = 4.1 μM*
IC50 = 1.8 μM*
* Growth inhibitory IC50 values for MCF7 human breast cancer cell lines
41Peyregne, V. P. et al. Mol. Cacncer Ther., 2005, 4, 595-602.
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Quinones as Inhibitors of Cdc25B
Naphthoquinones
O
O
R
R
OH O
O
R
R
OH
S
En
O
O
R
R
O
S
En
H
S En
Hydrogen bonding between the enolic anion and the hydroxy group
42Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
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NaphthoquinonesBinding Mode
Quinones as Inhibitors of Cdc25B
Ligand Ntot foccΔGbind
(kcal/mol)GOLD score
NSC 128981 11 11 -7.89 52.28
Result of 50 independent Autodock and GOLD docking runs –
NSC 128981IC50 = 0.62 μM
S
O
O
NH
O CH3
Cl
43Lavechhia, A. et al. Chem Med Chem, 2006,1, 540-550.
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Quinolinediones
Quinones as Inhibitors of Cdc25B
O
NNH
N
O
O
Cl
IC50 = 0.21 μM
NSC 663284
* in-vitro IC50 value
44Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
Inhibits enzyme in both reversible and irreversible manner.
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Chlorine moiety is not required
Quinolinediones
Quinones as Inhibitors of Cdc25B
IC50 = 0.21 μM
* in-vitro IC50 values
45Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
O
NNH
N
O
O
Cl
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Decreased activity when - substituted with different groups shifted to 6-position (IC50 = 20μM)
2-morpholin-4-ylethylamino moiety increases activity
Chlorine moiety is not required
O
NNH
N
O
O
Cl
Quinolinediones
Quinones as Inhibitors of Cdc25B
IC50 = 0.21 μM
* in-vitro IC50 values
45Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
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2-morpholin-4-ylethylamino moiety increases activity
Chlorine moiety is not required
O
NNH
N
O
O
Cl
R
Small groups are tolerated
Quinolinediones
Quinones as Inhibitors of Cdc25B
R = 2-Me : IC50 = 4.6 μM
R = 4-Me : IC50 = 4.6 μM
R = 2-CN : IC50 = 3.7 μM
N N
N
N
N
Aza analogues are less active
IC50 = 0.21 μM
* in-vitro IC50 values
45Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
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QuinolinedionesBinding Mode
Quinones as Inhibitors of Cdc25B
O
NNH
N
O
O
Cl
Ligand Ntot foccΔGbind
(kcal/mol)GOLD score
NSC 663284 10 26 -8.12 42.97
Result of 50 independent Autodock and GOLD docking runs –
NSC 663284IC50 = 0.21 μM
Two modes were observed – Autodock placed the
quinolinequinone ring into the “swimming pool” cavity
GOLD placed quinolinequinone ring into the catalytic site
46Lavechhia, A. et al. Chem Med Chem, 2006,1, 540-550.
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QuinolinedionesBinding Mode
Autodock GOLD
Quinones as Inhibitors of Cdc25B
47Lavechhia, A. et al. Chem Med Chem, 2006,1, 540-550.
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Indolyldihydroxyquinones
Quinones as Inhibitors of Cdc25B
O
O
OH
HO
HN
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.
48Sohn, J. et al. J. Med. Chem., 2003, 46, 2580-2588.
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Indolyldihydroxyquinones
Quinones as Inhibitors of Cdc25B
O
O
OH
HO
HN 2
45
6
7
Methyl group is tolerated
Substituents of size greater than propyl increase
potency
Halides and benzyloxy increase potency
Methyl is deleteriousIC50 = 18 μM
Substitution reduced activity
Substitution reduced activity
49Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
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Indolyldihydroxyquinones
Binding Mode
Quinones as Inhibitors of Cdc25B
Ligand Ntot foccΔGbind
(kcal/mol)GOLD score
Compound 1 11 11 -7.89 52.28
Result of 50 independent Autodock and GOLD docking runs –
O
O
OH
HO
HN
Compound 1IC50 = 1 μM
Two modes were observed – Autodock placed the quinone ring
into the “swimming pool” cavity
GOLD placed quinone ring into the catalytic site
50Lavechhia, A. et al. Chem Med Chem, 2006,1, 540-550.
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Quinones as Inhibitors of Cdc25B
Indolyldihydroxyquinones
Binding Mode
Autodock GOLD
51Lavechhia, A. et al. Chem Med Chem, 2006,1, 540-550.
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Benzothiazole- and Benzoxazole- diones
Quinones as Inhibitors of Cdc25B
X
N
O
O
R
R
RX = S,O
O
N
O
O
R
HN
N
R = EtR = Ph
IC50 = 0.15 to 0.44 μM
O
N
O
O
R
NH
NR = EtR = Ph
S
N
O
O
CH3
HN
N
IC50 = 0.25 μM
Irreversible inhibition
52Garuti, L. et al. Current Medicinal Chemistry, 2008, 15, 573-580.
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Acceptor H-bond group
HS
Cys473
Phe475
Arg544
NH
H2N NH
OH
Tyr428
Phe543
Thr547 CH3
OH
NH
Trp550
Arg482
HN
NH2HN
A B C
Swimming poolCatalytic pocket
Arg479
NHH2N
NH
Acceptor H-bond group
Pharmacophoric Model for Cdc25B Reversible Inhibition
53Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.
D
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Arg544
NH
H2N NH
Arg482
HN
NH2HN
A B C
Swimming poolCatalytic pocket
Pharmacophoric Model
Group B : Core structure, mostly quinone
OH
Tyr428
53Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.
D
Acceptor H-bond group
Acceptor H-bond group
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Phe543
Thr547 CH3
OH
NH
Trp550
A B C
Swimming poolCatalytic pocket
Pharmacophoric Model
Group A : A bulky aromatic system 53Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.
D
Acceptor H-bond group
Acceptor H-bond group
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HS
Cys473
Phe475A B C
Swimming poolCatalytic pocket
Arg479
NHH2N
NH
Pharmacophoric Model
Group C : An aromatic ring or acceptor H-bond group 53Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.
D
Acceptor H-bond group
Acceptor H-bond group
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HS
Cys473
Phe475
Arg544
NH
H2N NH
OH
Tyr428
Phe543
Thr547 CH3
OH
NH
Trp550
Arg482
HN
NH2HN
A B C
Swimming poolCatalytic pocket
Arg479
NHH2N
NH
Pharmacophoric Model
Linker : An alkylic chain of 3-4 units 53Lavecchia, A. et al. Anitcancer Agents in Medicinal Chemistry, 2008, 8, 843-856.
D
Acceptor H-bond group
Acceptor H-bond group
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Future Prospects
54
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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.
55Sohn, J. et al. PNAS, 2004, 101, 16437-16441.
Future Prospects
Substrate Recognition Site -
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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
56Sohn, J. et al. PNAS, 2004, 101, 16437-16441.Rudolph, J. Biochemistry, 2007, 46, 3595-3604.
Future Prospects
Substrate Recognition Site -
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57
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.
Cdc25B: magentaCdk2-pYpY–CycA : blue
Rudolph, J. Biochemistry, 2007, 46, 3595-3604.
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Future Prospects
A potential binding pocket
Binding of suitable ligandscould engage the substrates involved in substrate recognitionand interfere in enzyme/substrateassociation.
58
Arg 488
Arg 492
Tyr 497
Rudolph, J. Biochemistry, 2007, 46, 3595-3604.
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O O
NH
O
HN
COOH
O
NH
COOH
O
HN
O
NH
COOH
O
NH2
SO3H
Future Prospects
Peptide Derived Inhibitors
Inhibitors designed based on sequence homology with the protein substrate.
59Lazo, J. S. et al. Anticancer Agents in Medicinal Chemistry, 2008, 8, 837-842.
Active site peptide ligand
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60
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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CONCLUSIONS
61
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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ACKNOWLEDGEMENT
Dr. Glen Kellogg
Kellogg’s Molecular Modeling & Drug Design Group
Department of Medicinal Chemistry
62
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