cdc25 and cancer: molecular modelling approaches for identification of a chemical start point for...

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

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

Cdc25A

Cdk 2

Cdk 1

Cdk 4

Cdc25B Cdc25C

?

Cdk 2Cdc25A

Cdc25B

Cdc25C

60% identical over catalytic

domain

Cdc25 Phosphatases

Nature Reviews Cancer 7 (2007) 495-507

Misregulation in 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

Cdc25B

Cdc25C

Ras*

Ras*

Ras*

Causal relationship with cancer

Cancer Res 67 (2007) 6605-11

Causal relationship with cancer

• Over-expressed in many tumour types

• Acts as classical ‘co-operating’ oncogene

• Reduction inhibits cellular transformation

• Alternative to kinases

Cdc25 and cancer

Where do we start?

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

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

Quinone inhibitors vs standard treatment

Brezak et al, (2009), Int. J. Cancer, 124, 1449

Pancreatic Cancer xenograftsNo TreatmentVehicleIRC-083864 (i.v.)Gemcitabine (i.p.)

Initial approaches: modify existing reversible inhibitors

(1) Korean Patent

(3)Quinones

(2) Natural Product

Cdc25A IC50 >100 mM

Dysidiolide

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

Where did they come from?

(2)

(3)

Montes et al (2008), J. Chem. Inf. Model ,157 Brisson et al (2004), Mol. Pharm., 824

Physical screen

Total compounds tested

10,000

IC50 < 10 mM 23

Hit rate 0.23%

Virtual screen

Total compounds docked

310,000

Compounds tested 1,500

IC50 < 100 mM 11

Hit rate 0.73%

FRED, Surflex, LigandFit PRIME collection (ChemBridge)

Where do we start?

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)

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

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

PPACK

D-Phe-Pro-Arg-CH2Cl

BM14.1248

The 3D Field Overlay Principle

Add field points to each structure

Negative

Positive

Surface

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

Pairwise comparisons can pull out the common features of all three molecules

200 conformations

111 conformations

18 conformations

Energy cut-off 6 kcal/mol

Summarise common biology with field points

1 (conf 81)

2 (conf 5)

3 (conf 2)

Field point template (A)• Two other solutions identified

Defining virtual screening input

(2)(1) (3)

Template Compound 1 Compound 2 Compound 3

A 81 5 2

B 81 8 4

C 81 8 16

Fieldscreen Database

~100,000,000

List of commercially available compounds

High throughput virtual screening to identify novel series

1

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

20% hit rateNo structural similarity to any known actives.MW range 250-350

Including 3 from 1st list

(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

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

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:

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.

(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 ± 0.1

B 8.9 ± 0.5 17.2 ± 0.1

C 10.2 ± 0.3 47.3 ± 0.1

T5896241MW 337