“ Bromodomains : A new class of epigenetic targets ripe for small molecule drug discovery”

1. “Bromodomains: A new class of epigenetic targets ripe for small molecule drug discovery” Jason Witherington EpiNova DPU ELRIG – Manchester 2012

2. Outline • Brief introduction to Epigenetics • Luck strikes!.....discovery of small molecule bromodomain inhibitors • Exploiting serendipity through SBDD/FBDD • Brief overview of preclinical iBET biology

3. Epigenetics - Chromatin • DNA is packaged around histones and other proteins to form chromatin • Chromatin is highly dynamic material which undergoes remodelling to allow suppression or activation of genes • A number of Epigenetic mechanisms control chromatin remodelling including post-translational modifications (PTMs) on histone tails • Dysregulation of histonePTMs implicated in human disease

4. Epigenetics : Histone post-translational modifications PTM of histone tails • >70 sites are known mostly located in the unstructured N-terminal tails. • > 8 types of modification have been reported. • AA modified include : K, R, S, T, Y, H, E • Most of these are reversible and dynamic. • PTM rarely occur in isolation => complex pattern of modification = histone code. • Reader domains rarely occur in isolation • PTMs can have a direct impact on physical properties of individual nucleosomese.g. neutralisation of charge • PTM are recognised by specialised reader domains.

5. Apo-A1 phenotypic assay • Apo-A1 target for dyslipidemia • Upregulator reporter HTS identified several lead series including a BZD series. • Medicinal chemistry successfully optimised molecules to candidate selection without knowledge of molecular target. • Extensive profiling of compounds did not identify target for these molecules Chemoproteomics -1.4kb Firefly luciferase 5’-UTR ApoA1 Human ApoA1 promoter 3’-UTR ApoA1 Benzodiazepine

6. How were new medicines discovered ? 1999-2008 Between 1998-2008: • More first-in-class drugs were discovered by phenotypic screening • More follower drugs were discovered by target-based screening Nat. Rev Drug Discovery 10, 507 (2011)

7. Matrix Chemoproteomics – Overview of approach Biologically relevantsystem HepG2 & THP1 J Med Chem (2011) 54, 3827 Derivatised Compounds Active BZD Inactive BZD • Wash and Elution • Stringency • Compound / SDS PMM • Separate on 1-D Gel • active compound specific bands & low backgrounds PROTEIN IDENTITY LC/MS/MS I A

8. Compound Key RED = Active BLACK = Inactive BZD Inactive matrix BZD active matrix Matrix alone 200 kDa 150 100 75 50 40 30 20 Chemoproteomics BET (BromoDomain & Extra Terminal) proteins identified +Series Xinactive + Series X active + BZD inactive + BZD active kDa 200 150 100 75 50 40 30 20 All bands identified as BET family proteins Brd2, Brd3, Brd4 Competition experiments suggest that actives from BZD and other series specifically interact with BET proteins

9. Active BZD – 1mM BRD4 siRNA: 500nM -1.6 96hr 48hr -1.4 -1.2 -1 DCT -0.8 24hr 72hr -0.6 -0.4 48hr 24hr -0.2 1 0 0hr 0.2 Brd4 knockdown induces Apo-A1 upregulation • Apo-A1 activators are ligands for the BET proteins • Is this interaction responsible for Apo-A1 upregulation? • Increase in ApoA1 mRNA on addition of BZD • Increase in ApoA1 mRNA on BRD4 knockdown Increase in Apo-A1 mRNA

10. BET binding correlates with Apo-A1 cellular activity BZDs Apo-A1 pec170 BRD4 FP p IC50 • Theoretical difficulties in tackling epigenetic PPI were not realised • Many diverse and potent compound obtained using cellular activity to guide SAR.

11. Bromodomains bind to acetylated lysine residues

12. Challenges with targeting epigenetic readers • Reader domains often bind PTM weakly=> no hot spots? • Multi-valency of protein-protein interactions => Tethered ligands • MegaDalton protein-protein/DNA complexes => will inhibiting a single interaction be enough for biological efficacy? • If protein-protein inhibition is poorly tractable=> how tractable are targeting epigenetic readers? PREVIOUS PHARMACEUTICAL FOCUS ON EPIENZYMES NOT EPIREADERS

13. bromodomain1 bromodomain2 ET domain Ac Ac Ac BRDs control gene transcription • Transcriptional co-regulators involved in histone binding complexes • Brd4 binds to cdk9/cyclinT (pTef-B) to positively regulate RNA pol II mediated transcription at multiple promoters Pol II P pTef-B BRD BRD transcription Acetylated lysines on Histones within euchromatin • ApoA1 compounds bind to BET BUT where specifically do the compounds interact?

14. Western Blot: anti FLAG PD: BZD active kDa 1 2 3 4 220 • GFP control • Flag Brd2 FL • Flag Brd2 N • Flag Brd2 C • Hek293 cells 120 100 80 60 50 40 30 20 801 1 FL N 473 C Chemoproteomicsimplicate Bromodomain of Brd2, 3, 4 Bromodomain 1 Bromodomain2 ET domain   X X

15. INACTIVE X INACTIVE Y ACTIVE X ACTIVE Y Biophysical data demonstrates specific binding 1 Brd2(1-473) ACTIVE X INACTIVE X 0.8 0.6 ACTIVE Y INACTIVE Y normalised CD 0.4 • Tool compounds stabilise all Brd2 bromodomain constructs 0.2 • 0 40 50 60 Temp (oC) KD 225nM@25°C BRD2_1 KD <70nM@25 °C BRD2_2 • BZD tool binds both N and C-terminal domains but kinetics and affinity at 25°C are different for each

16. Brd2 67-200 1 Brd2 338-473 2 3 Brd2 67-200 Brd2 338-473 N-terminal bromodomain N-terminal bromodomain C-terminal bromodomain C-terminal bromodomain Isothermal Titration Calorimetry demonstrates specific binding to both BRDs  1:1 46nM(16°C)  1:1 52nM(26°C)  2:1 30nM(26°C)

17. I-BET762 is a highly selective inhibitor of BET bromodomains Tm profiling 5-7oC 1-3oC <1oC I-BET762

18. iBET Broader Selectivity Profiling • Inactiveagainst a wide range of proteins

19. Where do the compounds bind? • N-terminal bromodomain of Brd2 is typical helical structure • Their role is to recognise acetylated marks on histones and other proteins • Compounds shown to displace the tetraAcH4 peptide •  Antagonise protein-protein interaction FRET assay for displacement of tetraacetylated H4

20. First Small Molecule X-ray co-crystal confirms binding in the acetylated lysine pocket H4 peptide • Recognition of carbonyl of AcKpreserved (N156,Y113) • F-(VP)-Y-(CAS)-N AcK binding site • Common to 44 out of 58 bromodomains • H2O structure in pocket preserved. • NH interactions of AcKnot preserved

21. Interactions of BZD outside the AcKpocket BrdT – Nature (2009)

22. Bromodomains can deliver both probes and drug like molecules *3mg/kg p.o.;1mg/kg i.v.

23. Optimisation of dimethylisoxazole HTS lead to in vivo probe I-BET 151 HTS Lead I-BET 151 BMCL, 2012, 2963 BMCL, 2012, 2968

24. GSK525762 and GSK1210151 bind BET proteins using similar “hot spots” WPF ZA Channel I-BET 762 AcK pocket I-BET 151

25. Bromodomain Family and Structural Coverage BRPF1 BRD2_1 BRD3_1 BRD1 BRD3_2 CREBBP BRD4_1 BPRF3 BRD2_2 EP300 >50 bromodomains In isolation or combination with other domains Multiple opportunities for clinical utility BRD9 BRDT_1 BRDT_2 BRD4_2 BRD7 BAZ1A KIAA1240 ATAD2 BRD8 WDR9_2 PHIP_2 TAF1_1 BRWD3_2 BAZ1B PRKCBP1 TAF1L_1 TAF1_2 TAF1L_2 CECR2 FALZ BAZ2B GCN5L2 PCAF BAZ2A ZMYND11 T T T Y T Y Y Y MLL TRIM33 WDR9_1 TRIM28 TIF1 ASH1L BRWD3_1 TRIM66 SP110 PHIP_1 SP140 SP100 LOC93349 PB1_1 PB1_3 PB1_2 PB1_5 PB1_4 SMARCA4 SMARCA2 Structure known Atypical AcK Binding Residue

26. Across the family there is significant structural divergence outside of the AcK binding region ZA Loop BC Loop

27. Exploiting Structural Knowledge :Fragments – Generation of a Hit-ID platform for Bromodomains • Knowledge of key ligand-protein interactions derived from the Bet programme lead-like compounds • Generation of a pharmacophore model • Selection of a focussed screening set >20% inhib at 200uM • Confirmation of the binding mode using crystallography • Creation of a fragment toolchest that binds in the AcK recognition pocket of the bromodomain

28. Fragment based discovery >40 Fragments crystallised 1400 Fragments screened Key Structural waters identified Pharmacophore refined "Fragment-based discovery of bromodomain inhibitors part 1: Inhibitor Binding Modes and Implications for lead discovery Author(s): Chung, Dean, Woolven and Bamborough

29. Application of FBBD for Bromodomains WPF shelf Pharmacophore "Fragment-based discovery of bromodomain inhibitors part 2: optimization of phenylisoxazolesulfonamides“ Author(s): Bamborough, Paul; Diallo, Hawa; Goodacre, Jonathan; Gordon, Laurie; Lewis, Antonia; Seal, Jon; Wilson, David; Woodrow, Michael; Chung, Chun-wa ACCEPTED

30. Application of Encoded Library Technology (ELT) Structural knowledge Construction and screening of libraries L I B R A R I E S enrichment Identification of Features Exploitation of Screening output 1 2 3 targets Hits Screening tools Probes ELT hits against target 2 libraries

31. Preclinical Biology

32. Nodal AND gene specific intervention? IFNb TNFa IL-6 pI:C LPS I-BET TNFa X X IFNb IL6 unaffected blocked blocked

33. LPS drives recruitment of Brd4 to selective promoters Compounds prevent this recruitment and block transcriptional activation BET compound displaces BRD4 from IFNb and IL-6 promoters (ChIP) IFNb IL-6 BRD4 / H3 SorenBeinke

34. Targeted intervention by I-BET Primary response genes Secondary response genes Pol II H3K4m3 H3K9Ac CpG high H3K9m1/2/3

35. Use of Chemoproteomics for target class expansion • Pharma industry mainly reliant on recombinant platforms • Large screening panels required for selectivity profiling (human/rat etc) • Brds occur in isolation & combination with other domains • Protein complexes modify function • Different complexes may form under different activation states &/or different tissues • EpiNova-Cellzome alliance provides a complementary screening platform to address the above

36. BET interacting proteins: MS-proteomic analysis Triple purification strategy Acetylated H4 tail (K4,K8,K12) Ac Nature (2011) 478, 529 H4 proteins binding directly or indirectly to histone marks Antibody against BRD2/3/4 proteins binding directly or indirectly to I-BET • = BET inhibitor • (I-BET) BET protein imuno-complexes

37. BET interacting proteins: MS-proteomic analysis

38. I-BET762 is effective in multiple models of Multiple Myeloma log10 [I-BET762] [I-BET762] **

39. BET interacting proteins: MS-proteomic analysis

40. I-BET151 is a novel & selective inhibitor of BET proteins with improved PK properties I-BET151

41. I-BET151 has selectivity for MLL leukaemias

42. I-BET151 mediates disease control in MLL leukaemia models Transplant human MV411 leukaemia cells NOD-SCID Transplant syngeneic MLL-AF9 leukaemia cells C57BL/6

43. Summary • Chemoproteomics has been employed to identify a chemical opportunities against a previously intractable target class • Chemoproteomics has been utilised to allow the efficient selectivity profiling across the “Bromonome” using endogenous cell lysates • Chemoproteomics has demonstrated utility in defining clinical opportunities through complex identification

44. Effect of BET inhibition on LPS induced shock LPS 0h I-BET -1h I-BET 1.5h Nature, 468, p1119, 2010 therapeutic preventative

45. Summary….. • Use of chemoproteomics can be a powerful way to identify output of phenotypic screening • Previously “undruggable” reader class of epigenetic proteins are ripe for drug discovery • The iBETbromodomain family of proteins have profound preclinical biology (more this afternoon)

46. Kevin Lee