1. FGFR HGF Cdk6 Tom Blundell, Chris Abell, Ashok Venkitaraman, Alfonso Martinez Arias, Luca Pellegrini, Marko Hyvonen, Carol Robinson University of Cambridge BRCA2 R93 E115 H114 K91 S161 Q173 D171 Drug-like fragments Fragment- based approaches to the design of candidate drugs that interrupt protein-protein interactions involved in cell regulation

2. Many difficult targets are found amongst multiprotein complexes of cell signalling and regulation Further complication: often weak binary complexes lead to stable multiprotein complexes.

3. Target Identification & Validation Lead Development Lead Discovery Target Selection Traditional use of X-ray and modelled 3D structures ScreeningHits-to-leads Lead optimisation Sequence-structure homology recognition Structural genomics Structure-based Screening Structural biology and Drug Discovery

4. Complex of cyclin D-dependent kinase Cdk6 bound to the cell cycle inhibitor p19 INK4d. Brotherton, Dhanaraj, Blundell, Laue, Nature 395, 244-250, 1998 Large, flat and uninteresting: difficult to bind small molecules?

5. STRATEGIES FOR LEAD DISCOVERY WITH TARGETS THAT ARE MULTIPROTEIN COMPLEXES 1. 1.Exploitation of hot spots in protein-protein interaction surfaces. 2. 2.Formation of large aggregates of hydrophobic or amphipathic molecules – a specific mechanism of non- specific inhibition! 3. 3.Exploitation of allosteric effects. 2. 2.Fragment –based appoaches

6. Multiprotein Complexes Preformed globular structures One or more partners become structured on assembly Discontinuous epitopes Continuous epitopes + + + Preformed but adaptive globular structures

7. 1. 1.RAD51 is an essential enzyme in DNA repair. 2. 2.The breast cancer susceptibility protein BRCA2 controls RAD51 activity via a direct protein-protein interaction. 3. 3.Disruption of the BRCA2-RAD51 binding interface blocks RAD51 activity. 1. 1.RAD51 is an essential enzyme in DNA repair. 2. 2.The breast cancer susceptibility protein BRCA2 controls RAD51 activity via a direct protein-protein interaction. 3. 3.Disruption of the BRCA2-RAD51 binding interface blocks RAD51 activity.

9. Expression and purification of RAD51 for lead identification. 1.NMR (in vitro) 2.X-ray (in crystal) Screening:

10. Figure 10 a, 986677. b, 1014147. c, 302916. d, 300842. e, 984356. f, 977759. The compound 977759 would probably be more co-planar than is predicted in the docking solution, due to bond conjugation. a fe d c b

11. Alternative Fragment strategies Growing Linking or coupling

12. Thrombin fragment identification Novel hits S1 Neutral Fragment Novel binding sites S2-S4 Fragment S2/S4 fragment potency 10  M Linked S1 and S2/S4 fragment: new hybrid compound potency 200nM

13. Thrombin inhibition S2-pocket S4-pocket S1-pocket Melagatran Ximelagatran Exanta (AZ) To identify novel, non-peptidic inhibitors which lack strongly basic functionality

14. Fragment linking: thrombin inhibition S2-pocket S4-pocket S1-pocket

15. S1 Fragment hits S1-pocket S2-pocket S4-pocket IC 50 = 330  M

16. S1-pocket S2-pocket S4-pocket IC 50 = 300  M S2-S4 Ligands

17. S1-pocket S2-pocket S4-pocket IC 50 = 3.4 nM Fragment Linking theoretical values K d (additive) 100 nM K d (superadditive) 0.1 nM IC 50 = 300  M IC 50 = 330  M

18. Biophysical chemist Screen fragments against protein targets using some of the following techniques: (i) X-ray crystallography (ii) NMR spectroscopy (Water LOGSY) (iii) Isothermal titration calorimetry (iv) Surface plasmon resonance (v) Non-covalent mass spectrometry Have recently opened a new facility for Biological Chemistry which includes new ITC and SPR equipment. cocktail of three fragments 0.5 mM + enzyme 20  M fragment 1 only + enzyme 2 11 3 1

19. Dynamic combinatorial chemistry (DCC) Reactive fragmentsFragment combinations Fragment combination bound to protein target Detect product either by (i) (i)Stopping equilibration and identifying major species (ii) (ii)Direct observation by x-ray crystallography M. S. Congreve et al., Angew. Chemie Int. Ed., 2003, 42, 4479-82

20. Targeting the RAD51:BRCA2 interaction with small molecules

21. The RAD51:RAD51 and the RAD51:BRCA2 interactions use the same interface RAD51 filament BRCA2

22. Essential Phe and Ala side-chains PheHisThrAlaSerGly F-H-T-A-S-G

23. Lead identification 1. 1.Strategy: Find fragments that bind the Phe or Ala pockets; link them Screen fragment library by mass spec, SPR, etc 2. 2.Factors: Leads may have a high MW and/or a low affinity Kd of the BRC:RAD51 interaction ~1µM in preliminary SPR studies 3. Initial aim: Leads with Kd ~1-10nM & most ‘drug-like’ characteristics

24. Proof-of-concept & optimization studies for potential leads 1. 1.Inhibition of the BRC peptide-RAD51 interaction in ELISA or FP assays 2. 2. Inhibition of RAD51 focus formation after DNA damage 3. 3. In vitro sensitization of cell lines to DNA damaging agents Increasing potency

25. Potential clinical profiles Genetic & cell biological studies demonstrate that disruption of the BRCA2-RAD51 interaction - Suppresses DNA double-strand break repair by homologous recombination during G2 Sensitizes cells to DNA cross-linking agents, PARP inhibitors, and replication blockers So, potential clinical profiles could include - Combination with radiation or radiomimetics to overcome G2 resistance Combination with DNA cross-linking agents, PARP poisons or topoisomerase inhibitors

26. Parallel approaches to lead identification Fragment-based screens Lead ID, proof-of-concept studies Lead optimization Pre-clin development Conventional screens Synthetic libraries [MRCT] Natural product libraries [EU]

27. Research Scientist Appointments 1. 1.Medicinal Chemist. 2. 2.Biophysical Chemist 3. 3.Molecular biologist/biochemist 4. 4.X-ray crystallography 5. 5.Cell biologist for assays

28. Biophysical chemist Screen fragments against protein targets using some of the following techniques: (i) X-ray crystallography (ii) NMR spectroscopy (Water LOGSY) (iii) Isothermal titration calorimetry (iv) Surface plasmon resonance (v) Non-covalent mass spectrometry Have recently opened a new facility for Biological Chemistry which includes new ITC and SPR equipment. cocktail of three fragments 0.5 mM + enzyme 20  M fragment 1 only + enzyme 2 11 3 1

29. Synthetic organic chemist Preparation of fragment library (analysis of chemical structures, knowledge of reactivity, solubility etc) Strategy to build up from initial fragment hits to lead compounds (going from mM to nM) Preparation of successive generations of compounds as go from hits to leads. Compound synthesis, purification and characterisation (specific skil set, cannot be done by e.g. biochemist or molecular biologist) Development of fragment combining approaches (including dynamic combinatorial chemistry and dynamic combinatorial crystallography) We have a recent success at carrying out fragment based drug discovery in the University resulting in nM inhibitor (thrombin project)

30. Thrombin inhibition S2-pocket S4-pocket S1-pocket Melagatran Ximelagatran Exanta (AZ) To identify novel, non-peptidic inhibitors which lack strongly basic functionality

31. Fragment linking: thrombin inhibition S2-pocket S4-pocket S1-pocket

32. S1 Fragment hits S1-pocket S2-pocket S4-pocket IC 50 = 330  M

33. S1-pocket S2-pocket S4-pocket IC 50 = 300  M S2-S4 Ligands

34. S1-pocket S2-pocket S4-pocket IC 50 = 3.4 nM Fragment Linking theoretical values K d (additive) 100 nM K d (superadditive) 0.1 nM IC 50 = 300  M IC 50 = 330  M

35. Biophysical chemist Screen fragments against protein targets using some of the following techniques: (i) X-ray crystallography (ii) NMR spectroscopy (Water LOGSY) (iii) Isothermal titration calorimetry (iv) Surface plasmon resonance (v) Non-covalent mass spectrometry Have recently opened a new facility for Biological Chemistry which includes new ITC and SPR equipment. cocktail of three fragments 0.5 mM + enzyme 20  M fragment 1 only + enzyme 2 11 3 1

36. Notch is a single transmembrane receptor activated by ligands of the DSL family

37. Ligand interaction triggers a series if regulated proteolytic events wich release the intracellular domain of Notch to interacy with nuclear proteins in particular CSL  -secretase (PS)

38. Alterations in Notch activity lead to Alagile syndrome Spondylocostal dysostosis CADASIL and in many instances Notch plays a central role in most (all) cell fate decision during development and the differentiation of mES and hES cells Cancer

39. Grabher et al. Nature Reviews Cancer advance online publication; published online 13 April 2006 | doi:10.1038/nrc1880 T cell acute lymphoblastic leukemia (T-ALL)

40.  -secretase has other substrates Cadherin ErbB-4 ß-amyloid precursor protein (APP) Corollary: other processes might be affected  -secretase inhibits Notch activation and thus has secondary effects on Notch signalling in various places e.g intestine and the immune system In transgenic models induction of lymphomas through activation of Notch has a very long latency which is shortened by secondary events. The reverse is also truth e.g Ikaros-Notch connection Corollary: targetting Notch activation might not have an effect on endogenous Notch before being effective on the lymphoma. Corollary: one could use  -secretase inhibitors to curtail CRC. However at the moment this results in all cell differentiating and an dissappearance of the epithelium through exhaustion of the niche Notch

41. Grabher et al. Nature Reviews Cancer advance online publication; published online 13 April 2006 | doi:10.1038/nrc1880 T cell acute lymphoblastic leukemia (T-ALL)

43. Endocytosis Traffick Stability Transcription

44.  -secretase has other substrates Cadherin ErbB-4 ß-amyloid precursor protein (APP)  -secretase inhibits Notch activation and thus has secondary effects on Notch signalling in various places e.g intestine and the immune system Specificity: Notch ANK only interact with Notch effectors. This allows selective interference with Notch activity. Targetting Notch ANK will not affect Notch activation but rather active Notch. It will be possible to target specific processes e.g. transcription, traffick, degradation. It Will diminish interference with normal Notch signalling. In transgenic models induction of lymphomas through activation of Notch has a very long latency which is shortened by secondary events. The reverse is also truth e.g Ikaros-Notch connection Targetting Notch ANK will allow a particularly effective way of treating pathologies triggered by promiscuous transcriptional activity of Notch