Slide 1

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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 Biology
2Drug Discovery Centre
Cdc25 Phosphatases
Inactive Cdk
Cyc
PT
PY
Cdc25A
Cdk
ADP
Cdc25
Wee1
Cyc
P
P
T
Y
Cdk
Active Cdk
Cdc25B
ATP
Cdc25C
60% identical
over catalytic
domain
Cdc25 Phosphatases
Cdc25B
Cdc25C
Cdk 1
Cdc25A
Cdk 2
Cdc25B
Cdk 2
Cdk 4
Cdc25C
Cdc25A
?
60% identical
over catalytic
domain
Misregulation in cancer
Nature Reviews Cancer 7 (2007) 495-507
Causal relationship with cancer
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
Ras*
Cdc25B
Ras*
Cdc25C
Ras*
Causal relationship with cancer
Cancer Res 67 (2007) 6605-11
Cdc25 and cancer
•Over-expressed in many tumour types
•Acts as classical ‘co-operating’ oncogene
•Reduction inhibits cellular transformation
•Alternative to kinases
Where do we start?
Enzyme
structure
known?
Bioassays
available?
Any
known
ligands?
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.
Swimming pool
Catalytic site
CDK interaction site
Cdc25B: 1QB0.pdb
Quinones: irreversible Cdc25 inhibitors
Vitamin K3
BN82685
Cdc25B IC50 3.8mM
IRC-083864/Debio-0931
Cdc25A: 23 nM
Cdc25B: 26 nM
Cdc25C: 23 nM
Quinones arrest cell cycle by
•oxidation of Cys in catalytic site
•irreversible reaction with Cys
Quinone inhibitors vs standard treatment
Pancreatic Cancer xenografts
No Treatment
Vehicle
IRC-083864 (i.v.)
Gemcitabine (i.p.)
Brezak et al, (2009), Int. J. Cancer, 124, 1449
Initial approaches: modify existing reversible
inhibitors
(1) Korean Patent
(3)Quinones
(2) Natural Product
Dysidiolide
Cdc25A
IC50 >100 mM
Small set of reversible inhibitors known
(3)
PITT-9131
(1)
Assay
IC50 (mM)
Cdc25B
2.0
(2)
Kim et al WO2006/101307
Assay
IC50 (mM)
Cdc25A, B, C
5-10
Brisson et al (2004),
Mol. Pharm., 824
Assay
IC50 (mM)
MBP-Cdc25B3
13.0 ± 0.5
Montes et al (2008), J. Chem. Inf. Model ,157
Where did they come from?
(3)
(2)
Virtual screen
Physical screen
Total compounds
docked
310,000
Total compounds
tested
10,000
Compounds tested
1,500
IC50 < 10 mM
23
IC50 < 100 mM
11
Hit rate
0.23%
Hit rate
0.73%
FRED, Surflex, LigandFit
PRIME collection (ChemBridge)
Montes et al (2008), J. Chem. Inf. Model ,157
Brisson et al (2004), Mol. Pharm., 824
Where do we start?
Enzyme
structure
known?
Bioassays
available?
Any
known
ligands?
Modelling with field points
•
Virtual
Screen
De
Novo
Design
Scaffold
Hopping
• Problem 1
• Problem 2
Library
Design
- few known ligands
- no X-ray data (until
2007)
•
QSAR
Ligand based approach to find novel
antagonists for GPCRs
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)
Thrombin X-rays
PPACK
Proteins don’t
see ligands in
the same
way as
chemists
BM14.1248
PDB reference codes
PPACK: 1PPB
BM14.1248: 1UVT
Why do we need field points?
Thrombin
inhibitors
cLogP
BM14.1248
PPACK
D-Phe-Pro-Arg-CH2Cl
3.10
0.24
H-bond donors
2
5
H-bond acceptors
5
5
2D similarity
0.17 (Tanimoto)
The 3D Field Overlay Principle
Add field points to each structure
Negative
Surface
Positive
Shape
The 3D Field Overlay Principle
Compare individual sets of field points
The 3D Field Overlay Principle
The 3D Field Overlay Principle
rms fit to crystal structure 0.76
T.Cheeseright et al (2006),J. Chem. Inf. Mod., 665
Create new class of reversible Cdc25
inhibitor using field points
•Create single model from 3 different ligands
Model
•Dissect out field point pattern for one compound
•Use as pharmacophore probe for virtual screen
Virtual
Screen
•Hunt for compounds with similar field point patterns
•Purchase commercial compounds suggested
Test
•Test compounds in enzyme bioassay
Pairwise comparisons can pull out the common
features of all three molecules
Energy cut-off 6 kcal/mol
1
200 conformations
2
111
conformations
3
18
conformations
Summarise common biology with field points
1 (conf 81)
2 (conf 5)
Field point template (A)
•Two other solutions identified
3 (conf 2)
Defining virtual screening input
(1)
(2)
(3)
Template
Compound 1
Compound 2
Compound 3
A
81
5
2
B
81
8
4
C
81
8
16
High throughput virtual screening to identify
novel series
1
~100,000,000
Fieldscreen Database
List of commercially available compounds
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 …….
Processing the 2nd hitlist
100 compounds
40 available for
purchase
Including 3 from 1st list
No structural similarity to any
known actives.
MW range 250-350
35 arrived &
tested
7 active
(2050mM)
20% hit rate
Initial thiazole hits from virtual screen
Cdc25B IC50 2.3 mM
(1) MW 484
•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
T5896241
MW 337
Cdc25A IC50 35.5 ± 0.1 mM
Cdc25B IC50 17.2 ± 0.1 mM
Cdc25C IC50 47.3 ± 0.1 mM
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
Thanks to
James Collins
Michelle Heathcote
Hayley Cordingley
Cathy Tralau-Stewart
Albert Jaxa-Chamiec
Funding from:
Alan Armstrong
Katie Chapman
Kate Judd
Kathy Scott
Pascale Hazel
Andy Vinter
Mark Mackey
Tim Cheeseright
www.cresset-group.com
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.
Solubility is a problem with some initial hits
Cdc25B IC50 2.3 mM
No detergent
(1) MW 484
T5896241
MW 337
Cdc25
isoform
IC50 (uM)
No
detergent
N=3
IC50 (uM)
With
detergent
N=4-7
A
2.4 ± 0.3
35.5 ± 0.1
B
8.9 ± 0.5
17.2 ± 0.1
C
10.2 ± 0.3
47.3 ± 0.1
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