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, 41
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 binding1
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/min.kg
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