David Mann 1 & Caroline Low 2 1 Molecular Cell Biology 2 Drug Discovery Centre

1. 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

2. Cdc25 Phosphatases Inactive Cdk T Cdk Cyc Cyc Y P P P P ADP 60% identical over catalytic domain Wee1 ATP T Cdk Y Active Cdk Cdc25A Cdc25B Cdc25C Cdc25

3. Cdc25 Phosphatases Cdk 2 Cdk 2 Cdk 1 60% identical over catalytic domain Cdk 4 ? Cdc25A Cdc25B Cdc25C Cdc25A Cdc25B Cdc25C

4. Misregulation in cancer Nature Reviews Cancer 7 (2007) 495-507

5. 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. Ras* Ras* Ras* Cdc25A Cdc25B Cdc25C

6. Causal relationship with cancer Cancer Res 67 (2007) 6605-11

7. Cdc25 and cancer • Over-expressed in many tumour types • Acts as classical ‘co-operating’ oncogene • Reduction inhibits cellular transformation • Alternative to kinases

8. Where do we start?

9. 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 • nucleophilicreactivity of thethiolateanion of the catalytic cysteine residue. Swimming pool Catalytic site CDK interaction site Cdc25B: 1QB0.pdb

10. 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

11. 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

12. Initial approaches: modify existing reversible inhibitors (1) Korean Patent (2) Natural Product Dysidiolide (3)Quinones Cdc25A IC50 >100 mM

13. Small set of reversible inhibitors known PITT-9131 (1) (2) Brisson et al (2004), Mol. Pharm., 824 (3) Kim et al WO2006/101307 Montes et al (2008), J. Chem. Inf. Model ,157

14. Where did they come from? (2) (3) FRED, Surflex, LigandFit PRIME collection (ChemBridge) Montes et al (2008), J. Chem. Inf. Model ,157 Brisson et al (2004), Mol. Pharm., 824

15. Where do we start?

16. 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)

17. Thrombin X-rays Proteins don’t see ligands in the same way as chemists PPACK BM14.1248 PDB reference codes PPACK: 1PPB BM14.1248: 1UVT

18. PPACK D-Phe-Pro-Arg-CH2Cl Why do we need field points? Thrombin inhibitors BM14.1248

19. Negative Surface Positive Shape The 3D Field Overlay Principle • Add field points to each structure

20. The 3D Field Overlay Principle • Compare individual sets of field points

21. The 3D Field Overlay Principle

22. The 3D Field Overlay Principle rms fit to crystal structure 0.76 T.Cheeseright et al (2006),J. Chem. Inf. Mod., 665

23. Create new class of reversible Cdc25 inhibitor using field points

24. Pairwise comparisons can pull out the common features of all three molecules Energy cut-off 6 kcal/mol 200 conformations 18 conformations 111 conformations

25. Summarise common biology with field points 1 (conf 81) 2 (conf 5) • Field point template (A) • Two other solutions identified 3 (conf 2)

26. Defining virtual screening input (1) (2) (3)

27. Fieldscreen Database ~100,000,000 High throughput virtual screening to identify novel series 1 List of commercially available compounds

28. 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 …….

29. Processing the 2ndhitlist Including 3 from 1st list No structural similarity to any known actives. MW range 250-350 20% hit rate

30. Initial thiazole hits from virtual screen Cdc25B IC50 2.3 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 (1) MW 484 T5896241 MW 337 Cdc25A IC50 35.5 ± 0.1 mM Cdc25B IC50 17.2 ± 0.1 mM Cdc25C IC50 47.3 ± 0.1 mM

31. Summary of project to date • Created single model from three different chemotypes with FieldTemplater • Identified bioactive conformations • Used one field point pattern as probe for virtual screen (FieldScreen) • Found compounds active in vitro at mM concentrations • Identified new chemotype for Cdc25 inhibitors • 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

32. Andy Vinter Mark Mackey Tim Cheeseright www.cresset-group.com Thanks to James Collins Alan Armstrong Michelle Heathcote Katie Chapman Hayley Cordingley Kate Judd Cathy Tralau-Stewart Kathy Scott Albert Jaxa-ChamiecPascale Hazel Funding from:

33. 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.

34. Solubility is a problem with some initial hits Cdc25B IC50 2.3 mM No detergent (1) MW 484 T5896241 MW 337