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

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Presentation on theme: "Bromodomains: A new class of epigenetic targets ripe for small molecule drug discovery Jason Witherington EpiNova DPU ELRIG – Manchester 2012."— Presentation transcript:

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 histone PTMs implicated in human disease

4 Epigenetics : Histone post-translational modifications PTMs can have a direct impact on physical properties of individual nucleosomes e.g. neutralisation of charge PTM are recognised by specialised reader domains. 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

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 Benzodiazepine 5-UTR ApoA1 3-UTR ApoA1 -1.4kb Human ApoA1 promoter Firefly luciferase

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

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

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

9 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 Active BZD – 1mMBRD4 siRNA: 500nM DCT 24hr 48hr 96hr 0hr 24hr 48hr 72hr Increase in Apo-A1 mRNA

10 BET binding correlates with Apo-A1 cellular activity BZDs BRD4 FP p IC50 Apo-A1 pec170 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 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 ET domain bromodomain2bromodomain1 Ac Acetylated lysines on Histones within euchromatin Pol II transcription BRD pTef-B P ApoA1 compounds bind to BET BUT where specifically do the compounds interact?

14 Chemoproteomics implicate Bromodomain of Brd2, 3, 4 Bromodomain 1Bromodomain 2 ET domain X kDa 1.GFP control 2.Flag Brd2 FL 3.Flag Brd2 N 4.Flag Brd2 C Hek293 cells 1234 PD: BZD active Western Blot: anti FLAG FL N C X

15 Biophysical data demonstrates specific binding K D BRD2_1 K D °C BRD2_2 BZD tool binds both N and C-terminal domains but kinetics and affinity at 25°C are different for each normalised CD Brd2(1-473) Temp ( o C) INACTIVE X INACTIVE Y ACTIVE X ACTIVE Y T ool compounds stabilise all Brd2 bromodomain constructs ACTIVE XINACTIVE X ACTIVE YINACTIVE Y

16 Isothermal Titration Calorimetry demonstrates specific binding to both BRDs C-terminal bromodomain N-terminal bromodomain Brd Brd C-terminal bromodomain N-terminal bromodomain 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 5-7 o C 1-3 o C <1 o C I-BET762

18 iBET Broader Selectivity Profiling Inactive against 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 AcK preserved (N156,Y113) F-(VP)-Y-(CAS)-N AcK binding site Common to 44 out of 58 bromodomains H 2 O structure in pocket preserved. NH interactions of AcK not preserved

21 Interactions of BZD outside the AcK pocket BrdT – Nature (2009)

22 Bromodomains can deliver both probes and drug like molecules iBET 762 clogP, PSA, MWt~2, ~80, ~400 BRD2/3/4 pIC /6.7/6.7 hERG EC 50 Ion Works (Dof)100uM Patch Express61uM Rat (Mouse) PK* Clb (mL/min/Kg); Vss (L/kg); t½ (h), %Fpo 63 (24), 1.8 (1.7), 0.5 (0.8), 27 (22) Dog PK*5, 1.8, 5.9, 44 Unbound fraction in blood (R/D/Mou/H)0.18 /0.24/0.21/0.19 CYP inhibition IC 50 s (uM)> 33 P450 TDI<2-fold *3mg/kg p.o.; 1mg/kg i.v.

23 Optimisation of dimethyl isoxazole HTS lead to in vivo probe I-BET 151 CLi microsomes (mL/min.Kg) CLb ml/min.kgVd L/kgT½ hF % Rat< Dog minipig< Human1.1 BMCL, 2012, 2963 BMCL, 2012, 2968 HTS Lead I-BET 151

24 GSK and GSK bind BET proteins using similar hot spots WPF ZA Channel AcK pocket I-BET 762 I-BET 151

25 Bromodomain Family and Structural Coverage BRPF1 BRD1 BPRF3 BRD9 BRD7 KIAA1240 ATAD2 WDR9_2 PHIP_2 BRWD3_2 BAZ2B BAZ2A ZMYND11 CREBBP EP300 BAZ1A BRD8 BRDT_1 BRD4_1 BRD2_1 BRD3_1 BRD3_2 BRDT_2 BRD2_2 BRD4_2 TAF1_1 TAF1L_1 TAF1_2 TAF1L_2 PRKCBP1 CECR2 FALZ GCN5L2 PCAF WDR9_1 BRWD3_1 PHIP_1 ASH1L PB1_1 PB1_3 PB1_2 PB1_4 PB1 _ 5 SMARCA2 SMARCA4 TRIM33 TIF1 TRIM66 MLL SP110 SP100 LOC93349 SP140 TRIM28 BAZ1B Structure known Atypical AcK Binding Residue T T T T Y Y Y Y >50 bromodomains In isolation or combination with other domains Multiple opportunities for clinical utility

26 Across the family there is significant structural divergence outside of the AcK binding region BC Loop ZA 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 Creation of a fragment toolchest that binds in the AcK recognition pocket of the bromodomain Confirmation of the binding mode using crystallography >20% inhib at 200uM

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

29 pIC 50 BRD 2 pIC 50 BRD 3pIC 50 BRD 4 < 4.0 < 4.0 (LE< 0.43) pIC 50 PBMC TNF < 4.7 pIC 50 BRD 2 pIC 50 BRD 3pIC 50 BRD (LE 0.38) pIC 50 PBMC TNF 6.5 Pharmacophore WPF shelf Application of FBBD for Bromodomains "Fragment-based discovery of bromodomain inhibitors part 2: optimization of phenylisoxazole sulfonamides 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) LIBRARIES LIBRARIESLIBRARIES TARGETS Structural knowledgeConstruction and screening of libraries Identification of Features ELT hits against target 2 Exploitation of Screening output 1.Hits 2.Screening tools 3.Probes

31 Preclinical Biology

32 Nodal AND gene specific intervention? X X pI:CLPS TNFa IL6 IFNb I-BET unaffectedblocked TNFaIL-6 IFNb

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

34 Targeted intervention by I-BET Secondary response genesPrimary response genes CpG high H3K4m3 H3K9Ac Pol II 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 proteins binding directly or indirectly to I-BET proteins binding directly or indirectly to histone marks BET protein imuno- complexes = BET inhibitor (I-BET) Ac Acetylated H4 tail (K4,K8,K12) H4 Antibody against BRD2/3/4 Triple purification strategy BET interacting proteins: MS-proteomic analysis Nature (2011) 478, 529

37 BET interacting proteins: MS-proteomic analysis

38 I-BET762 is effective in multiple models of Multiple Myeloma ** [I-BET762] log 10 [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 NOD-SCID Transplant human MV411 leukaemia cells 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 0h LPS therapeutic I-BET 1.5h preventative I-BET -1h Nature, 468, p1119, 2010

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 iBET bromodomain family of proteins have profound preclinical biology (more this afternoon)

46 Kevin Lee

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