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Jason Witherington EpiNova DPU

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

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
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 nucleosomes e.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 5’-UTR ApoA1 3’-UTR -1.4kb Human ApoA1 promoter Firefly luciferase Benzodiazepine

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

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

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 Increase in Apo-A1 mRNA Active BZD – 1mM BRD4 siRNA: 500nM DCT 24hr 48hr 96hr 0hr 72hr -1.6 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0.2 1 We were therefore pretty convinced that the Apo-A1 activators were ligands for the Bet proteins. Which leads naturally to the question is this the interaction that is responsible for Apo-A1 upregulation? The histogram below shows in blue that application of the BZD cmpd to HepG2 cells results in an increase in Apo-A1 mRNA. In red you can see that this response can be induced using siRNA knockdown of Brd4. Clearly linking this BET protein with Apo-A1.

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 BRDs control gene transcription
ET domain bromodomain2 bromodomain1 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 pTef-B P transcription BRD Ac Ac Ac Acetylated lysines on Histones within euchromatin ApoA1 compounds bind to BET BUT where specifically do the compounds interact?

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

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

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

17 I-BET762 is a highly selective inhibitor of BET bromodomains
Tm profiling 5-7oC 1-3oC <1oC I-BET762 Not all protein-protein are difficult. This protein surface is filled with crevices and pockets. A yellow histone peptide binds along one of these with the Ac-K buried deep into a recognition pocket. Overlaying this with the white BZD compound shows that points into the Ac-K whilst this runs along the peptide grove and this pendant benzyl group binds in a hydrophobic pocket that is not occupied by the peptide Overlaying this with the purple ATAQ compound shows a similar heterocyle in the Ac-K pocket and the compound now occupying space to the left of the natural peptide ligand Overlay it with the green isoxazole compound shows that this compound doesn’t really use the peptide groove at all and the isoxazole is the moiety occupying the Ac-K pocket

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 H2O 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 pIC50 6.8/6.7/6.7 hERG EC50 Ion Works (Dof) 100uM Patch Express 61uM 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 IC50s (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/ Vd L/kg T½ h F % Rat <0.53 18 2.1 1.7 66 Dog 17 38 3.0 1.2 16 minipig 15 1.6 65 Human 1.1 BMCL, 2012, 2963 BMCL, 2012, 2968

24 GSK525762 and GSK1210151 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 BRD2_1 BRD3_1 BRD1 CREBBP BRD3_2 BRD4_1 BPRF3 EP300 BRD2_2 BRD9 BRDT_1 >50 bromodomains In isolation or combination with other domains Multiple opportunities for clinical utility BRDT_2 BRD7 BRD4_2 BAZ1A KIAA1240 ATAD2 BRD8 WDR9_2 PHIP_2 BRWD3_2 TAF1_1 BAZ1B PRKCBP1 TAF1L_1 TAF1_2 CECR2 TAF1L_2 FALZ BAZ2B GCN5L2 BAZ2A PCAF ZMYND11 TRIM33 MLL T T TRIM28 WDR9_1 TIF1 Y T TRIM66 SP110 Y ASH1L BRWD3_1 Y PHIP_1 SP140 T SP100 LOC93349 PB1_1 Y PB1_3 PB1_5 PB1_2 SMARCA4 PB1_4 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 Confirmation of the binding mode using crystallography >20% inhib at 200uM Creation of a fragment toolchest that binds in the AcK recognition pocket of the bromodomain

28 Fragment based discovery
1400 Fragments screened >40 Fragments crystallised 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
pIC50 BRD 2 pIC50 BRD 3 pIC50 BRD 4 < 4.0 < 4.0 (LE< 0.43) pIC50 PBMC TNF < 4.7 WPF shelf Pharmacophore "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 pIC50 BRD 2 pIC50 BRD 3 pIC50 BRD 4 5.2 5.9 5.6 (LE 0.38) pIC50 PBMC TNF 6.5

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

31 Preclinical Biology

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

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

34 Targeted intervention by I-BET
Primary response genes Secondary 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 Triple purification strategy
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 = BET inhibitor (I-BET) Antibody against BRD2/3/4 proteins binding directly or indirectly to 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

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
preventative I-BET -1h therapeutic I-BET 1.5h LPS 0h 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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