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