David Mann 1 & Caroline Low 2 1 Molecular Cell Biology 2 Drug Discovery Centre
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Cdc25 and cancer: molecular modelling approaches for identification of a chemical start
point for drug discovery
David Mann1 & Caroline Low2
1Molecular Cell Biology2Drug Discovery Centre
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Cdk
Cyc
Cdk
Inactive Cdk
Active Cdk
Cdc25
P
PP
P TY
TY
ATP
Wee1
ADP
Cdc25A
Cdc25B
Cdc25C
60% identical over catalytic
domain
Cdc25 Phosphatases
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Cdc25A
Cdk 2
Cdk 1
Cdk 4
Cdc25B Cdc25C
?
Cdk 2Cdc25A
Cdc25B
Cdc25C
60% identical over catalytic
domain
Cdc25 Phosphatases
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Nature Reviews Cancer 7 (2007) 495-507
Misregulation in cancer
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Cdc25 phosphatases as potential human oncogenes.Galaktionov K, Lee AK, Eckstein J, Draetta G, Meckler J, Loda M & Beach D.Science 269 (1995) 1575-7.
Cdc25A
Cdc25B
Cdc25C
Ras*
Ras*
Ras*
Causal relationship with cancer
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Cancer Res 67 (2007) 6605-11
Causal relationship with cancer
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• Over-expressed in many tumour types
• Acts as classical ‘co-operating’ oncogene
• Reduction inhibits cellular transformation
• Alternative to kinases
Cdc25 and cancer
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Where do we start?
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Swimming pool
CDK interaction site
Catalytic site
What do we know about the structure of Cdc25
Structure-based design of Cdc25 inhibitors hampered by
• shallow active site region exposed to bulk solvent
• nucleophilic reactivity of the thiolate anion of the catalytic cysteine residue.
Cdc25B: 1QB0.pdb
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Quinones: irreversible Cdc25 inhibitors
BN82685Cdc25B IC50 3.8mM
IRC-083864/Debio-0931Cdc25A: 23 nMCdc25B: 26 nMCdc25C: 23 nM
Quinones arrest cell cycle by•oxidation of Cys in catalytic site•irreversible reaction with Cys
Vitamin K3
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Quinone inhibitors vs standard treatment
Brezak et al, (2009), Int. J. Cancer, 124, 1449
Pancreatic Cancer xenograftsNo TreatmentVehicleIRC-083864 (i.v.)Gemcitabine (i.p.)
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Initial approaches: modify existing reversible inhibitors
(1) Korean Patent
(3)Quinones
(2) Natural Product
Cdc25A IC50 >100 mM
Dysidiolide
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Small set of reversible inhibitors known
(2)
(1)
(3)
Assay IC50 (mM)MBP-Cdc25B3 13.0 ± 0.5
Montes et al (2008), J. Chem. Inf. Model ,157
Assay IC50 (mM)Cdc25B 2.0
Kim et al WO2006/101307
Assay IC50 (mM)Cdc25A, B, C 5-10
Brisson et al (2004), Mol. Pharm., 824
PITT-9131
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Where did they come from?
(2)(3)
Montes et al (2008), J. Chem. Inf. Model ,157 Brisson et al (2004), Mol. Pharm., 824
Physical screenTotal compounds tested
10,000
IC50 < 10 mM 23
Hit rate 0.23%
Virtual screenTotal compounds docked
310,000
Compounds tested 1,500
IC50 < 100 mM 11
Hit rate 0.73%
FRED, Surflex, LigandFit PRIME collection (ChemBridge)
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Where do we start?
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Modelling with field points
• Ligand based approach to find novel antagonists for GPCRs
• Problem 1 - few known ligands
• Problem 2 - no X-ray data (until 2007)
• Collaboration with Andy Vinter at James Black Foundation
• 3 clinical candidates developed with this approach
• 2002 Cresset founded to exploit virtual screening (www.cresset-group.com)
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Thrombin X-rays
PPACK
BM14.1248
PDB reference codes PPACK: 1PPB BM14.1248: 1UVT
Proteins don’t see ligands in the same way as chemists
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Why do we need field points?
cLogP 3.10 0.24
H-bond donors 2 5
H-bond acceptors 5 5
2D similarity 0.17 (Tanimoto)
Thrombin inhibitors
PPACKD-Phe-Pro-Arg-CH2Cl
BM14.1248
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The 3D Field Overlay Principle
Add field points to each structure
Negative
Positive
Surface
Shape
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The 3D Field Overlay Principle
Compare individual sets of field points
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The 3D Field Overlay Principle
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The 3D Field Overlay Principle
rms fit to crystal structure 0.76
T.Cheeseright et al (2006),J. Chem. Inf. Mod., 665
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Create new class of reversible Cdc25 inhibitor using field points
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Pairwise comparisons can pull out the common features of all three molecules
200 conformations
111 conformations
18 conformations
Energy cut-off 6 kcal/mol
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Summarise common biology with field points
1 (conf 81)
2 (conf 5)
3 (conf 2)
Field point template (A)• Two other solutions identified
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Defining virtual screening input
(2)(1) (3)
Template Compound 1 Compound 2 Compound 3A 81 5 2
B 81 8 4
C 81 8 16
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Fieldscreen Database
~100,000,000
List of commercially available compounds
High throughput virtual screening to identify novel series
1
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Fieldscreen results
• First screen gave trivial analogues of seed•Top 200 were analogues of Compound 1•989/1000 were pyrazoles
• So ran screen again WITHOUT pyrazoles in Fieldscreen database
• This time chose top 100 hits …….
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Processing the 2nd hitlist
20% hit rateNo structural similarity to any known actives.MW range 250-350
Including 3 from 1st list
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(1) MW 484
Cdc25B IC50 2.3 mM
Initial thiazole hits from virtual screen
T5896241MW 337
Cdc25A IC50 35.5 ± 0.1 mMCdc25B IC50 17.2 ± 0.1 mMCdc25C IC50 47.3 ± 0.1 mM
• Selective against related phosphatases•PTP1B, MKP-1 & 3 and alkaline phosphatases
• Cellular target confirmed (n=1)•predicted increase in phosphorylated CDK2
•Later compounds amongst most potent reversible Cdc25 inhibitors described
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Summary of project to date
1. Created single model from three different chemotypes with FieldTemplater
2. Identified bioactive conformations
3. Used one field point pattern as probe for virtual screen (FieldScreen)
4. Found compounds active in vitro at mM concentrations
5. Identified new chemotype for Cdc25 inhibitors
6. Series under development• Composition of matter patent filed• Synthesis of analogues underway to explore SAR• In vitro enzyme assay in place• Cell proliferation assays in place
31
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Thanks to
Andy VinterMark Mackey
Tim Cheeseright
www.cresset-group.com
James Collins Alan ArmstrongMichelle Heathcote Katie ChapmanHayley Cordingley Kate JuddCathy Tralau-Stewart Kathy ScottAlbert Jaxa-Chamiec Pascale Hazel
Funding from:
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Figure 5. From (1) Brezak et al, (2009), Int. J. Cancer, 124, 1449-1456) Growth inhibition of xenografted tumors in nude mice treated with IRC-083864. (a) Cells of the human pancreatic carcinoma cell line MIA PaCa-2 were injected subcutaneously into the flank of female athymic mice. Tumors were allowed to reach a volume of 100 mm3. Once tumors were established, treatment was started by intravenous route as 10 mg/kg once a week for 4 weeks (qwk × 4). Gemcitabine was used as current standard treatment. (b) Cells of the human prostate carcinoma cell line LNCaP were injected subcutaneously into the flank of female athymic mice. Tumors were allowed to reach a volume of 150 mm3. Once tumors were established, treatment was started by the oral route at 70 mg/kg for 2 days on /5 days off/ 2 on / 5 off /1 on. Paclitaxel (20 mg/kg, qodx5, iv) was used as current standard care.
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(1) MW 484
Cdc25B IC50 2.3 mMNo detergent
Solubility is a problem with some initial hits
Cdc25 isoform
IC50 (uM) No detergentN=3
IC50 (uM) With detergentN=4-7
A 2.4 ± 0.3 35.5 ± 0.1B 8.9 ± 0.5 17.2 ± 0.1C 10.2 ± 0.3 47.3 ± 0.1
T5896241MW 337