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Cdc25 and cancer: molecular modelling approaches for identification of a chemical start point for drug discovery David Mann 1 & Caroline Low 2 1 Molecular.

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Presentation on theme: "Cdc25 and cancer: molecular modelling approaches for identification of a chemical start point for drug discovery David Mann 1 & Caroline Low 2 1 Molecular."— Presentation transcript:

1 Cdc25 and cancer: molecular modelling approaches for identification of a chemical start point for drug discovery David Mann 1 & Caroline Low 2 1 Molecular Cell Biology 2 Drug Discovery Centre

2 Cdc25A Cdc25B Cdc25C 60% identical over catalytic domain Cdc25 Phosphatases

3 Cdc25A Cdk 2 Cdk 1 Cdk 4 Cdc25B Cdc25C ? Cdk 2 Cdc25ACdc25BCdc25C 60% identical over catalytic domain Cdc25 Phosphatases

4 Nature Reviews Cancer 7 (2007) Misregulation in cancer

5 Cdc25 phosphatases as potential human oncogenes. Galaktionov K, Lee AK, Eckstein J, Draetta G, Meckler J, Loda M & Beach D. Science 269 (1995) Cdc25A Cdc25B Cdc25C Ras* Causal relationship with cancer

6 Cancer Res 67 (2007) Causal relationship with cancer

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

8 Where do we start? Enzyme structure known? Bioassays available? Any known ligands?

9 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

10 Quinones: irreversible Cdc25 inhibitors BN82685 Cdc25B IC  M IRC /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 Vitamin K3

11 Quinone inhibitors vs standard treatment Brezak et al, (2009), Int. J. Cancer, 124, 1449 Pancreatic Cancer xenografts No Treatment Vehicle IRC (i.v.) Gemcitabine (i.p.) Pancreatic Cancer xenografts No Treatment Vehicle IRC (i.v.) Gemcitabine (i.p.)

12 Initial approaches: modify existing reversible inhibitors (1) Korean Patent (3)Quinones (2) Natural Product Cdc25A IC 50 >100 M Dysidiolide

13 Small set of reversible inhibitors known (2)(2) (1)(1) (3)(3) Assay IC50 (  M) MBP-Cdc25B313.0 ± 0.5 Montes et al (2008), J. Chem. Inf. Model,157 Assay IC50 (  M) Cdc25B2.0 Kim et al WO2006/ Assay IC50 (  M) Cdc25A, B, C5-10 Brisson et al (2004), Mol. Pharm., 824 PITT-9131

14 Where did they come from? (2)(2) (3)(3) Montes et al (2008), J. Chem. Inf. Model,157Brisson et al (2004), Mol. Pharm., 824 Physical screen Total compounds tested 10,000 IC 50 < 10  M 23 Hit rate0.23% Virtual screen Total compounds docked 310,000 Compounds tested1,500 IC 50 < 100  M 11 Hit rate0.73% FRED, Surflex, LigandFitPRIME collection (ChemBridge)

15 Where do we start? Enzyme structure known? Bioassays available? Any known ligands?

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) Virtual Screen Library Design QSAR Scaffold Hopping De Novo Design

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

18 Why do we need field points? Thrombin inhibitors PPACK D-Phe-Pro-Arg-CH 2 Cl BM

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

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

21 The 3D Field Overlay Principle

22 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 Model Create single model from 3 different ligands Dissect out field point pattern for one compound Virtual Screen Use as pharmacophore probe for virtual screen Hunt for compounds with similar field point patterns Test Purchase commercial compounds suggested Test compounds in enzyme bioassay

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

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

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

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

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 2 nd hitlist 100 compounds 40 available for purchase 35 arrived & tested 7 active (  M) 20% hit rate No structural similarity to any known actives. MW range Including 3 from 1 st list

30 (1) MW 484 Cdc25B IC  M Initial thiazole hits from virtual screen T MW 337 Cdc25A IC ± 0.1  M Cdc25B IC ± 0.1  M Cdc25C IC ± 0.1  M 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

31 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  M 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

32 Thanks to Andy Vinter Mark Mackey Tim Cheeseright James Collins Alan Armstrong Michelle HeathcoteKatie Chapman Hayley CordingleyKate Judd Cathy Tralau-StewartKathy Scott Albert Jaxa-ChamiecPascale Hazel James Collins Alan Armstrong Michelle HeathcoteKatie Chapman Hayley CordingleyKate Judd Cathy Tralau-StewartKathy Scott Albert Jaxa-ChamiecPascale Hazel Funding from:

33 Figure 5. From (1) Brezak et al, (2009), Int. J. Cancer, 124, ) Growth inhibition of xenografted tumors in nude mice treated with IRC (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 (1) MW 484 Cdc25B IC  M No detergent Solubility is a problem with some initial hits Cdc25 isoform IC 50 (uM) No detergent N=3 IC 50 (uM) With detergent N=4-7 A2.4 ± ± 0.1 B8.9 ± ± 0.1 C10.2 ± ± 0.1 T MW 337


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