“Bromodomains: A new class of epigenetic targets
ripe for small molecule drug discovery”
ELRIG – Manchester 2012
Jason Witherington
EpiNova DPU
Outline
 Brief introduction to Epigenetics
 Luck strikes!.....discovery of small molecule
bromodomain inhibitors
 Exploiting serendipity through SBDD/FBDD
 Brief overview of preclinical iBET biology
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
Epigenetics : Histone post-translational modifications
PTM of histone tails
• >70 sites are known mostly located in the unstructured Nterminal 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.
Apo-A1 phenotypic assay
• Apo-A1 target for dyslipidemia
• Upregulator reporter HTS identified several lead
series including a BZD series.
N
N
-1.4kb
N
Firefly luciferase
R2
N
Human ApoA1 promoter
5’-UTR
ApoA1
3’-UTR
ApoA1
R1
Benzodiazepine
• 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
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)
Chemoproteomics – Overview of approach
Biologically
relevant
system
HepG2 & THP1
J Med Chem (2011) 54, 3827
Wash and Elution
• Stringency
• Compound / SDS
Matrix
Derivatised
Compounds
Active BZD
Inactive BZD
N
N
N
N
N
N
R2
R2
N
N
R1
R1
PMM
Separate on 1-D Gel
• active compound
specific bands & low
backgrounds
PROTEIN
IDENTITY
LC/MS/MS
A
I
+ Series X
active
+Series X
inactive
+ BZD inactive
+ BZD active
Matrix alone
BZD active
matrix
BZD Inactive
matrix
Chemoproteomics BET (BromoDomain & Extra
Terminal) proteins identified
Compound Key
RED = Active
BLACK = Inactive
kDa
200
150
200 kDa
150
100
75
50
100
75
50
40
40
30
20
All bands identified as
BET family proteins
Brd2, Brd3, Brd4
30
20
Competition experiments suggest that actives
from BZD and other series specifically interact
with BET proteins
Brd4 knockdown induces Apo-A1 upregulation
 Apo-A1 activators are ligands for the BET proteins
Active BZD – 1mM
BRD4 siRNA: 500nM
-1.6
-1.4
96hr
48hr
-1.2
-1
DCT
Increase in Apo-A1 mRNA
 Is this interaction responsible for Apo-A1 upregulation?
– Increase in ApoA1 mRNA on addition of BZD
– Increase in ApoA1 mRNA on BRD4 knockdown
-0.8
24hr
-0.6
72hr
-0.4
-0.2
1
0
0.2
0hr
24hr 48hr
BET binding correlates with Apo-A1 cellular activity
Apo-A1 pec170
BZDs
N
N
N
N
N
O
O
Cl
I-BET762
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.
Bromodomains bind to acetylated lysine residues
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
BRDs control gene transcription
bromodomain1
bromodomain2
ET domain

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
Ac
transcription
BRD
Ac
Ac
Acetylated lysines on Histones within euchromatin
 ApoA1 compounds bind to BET BUT where
specifically do the compounds interact?
Chemoproteomics implicate Bromodomain of
Brd2, 3, 4
1
FL
N
C
Bromodomain 1
Bromodomain 2


X
ET domain
473
Western Blot: anti FLAG
PD: BZD active
1 2 3 4
kDa
220
120
100
80
X
60
50
40
30
20
1.
GFP control
2.
Flag Brd2 FL
3.
Flag Brd2 N
4.
Flag Brd2 C
Hek293 cells
801
Biophysical data demonstrates specific binding
Some BIAcore Results - GW842819X BZD active
N
1
Brd2(1-473)
Brd2 67-200
60
40
ACTIVE X
80
O
INACTIVE
X
INACTIVE Y
60
0.4
10
R esp o nse
KD = 2.25e-7M
Rmax = 41 => 40% surface
is active.
20
KD < 7.0e-8M
Rmax = 74 => 70% surface
isool
active.compounds stabilise
koff ~ 0.01s-1 ± 0.03 s-1constructs
bromodomain
kon
40
T
20
0
0
•0
1e-9
N
GW841819X
ACTIVE Y
30
-10
N
100
ACTIVE Y
0.2
O
U
120
ACTIVE X
0.6
N
N
Steady State: Fc=1 Spot=2-r corr
RU
INACTIVE Y
50
R es p on se
normalised CD
INACTIVE X
Steady State: Fc=1 Spot=1-r corr
RU
0.8
Brd2 338-473
40
1e-8
1e-7
1e-6
Conc
50
Temp
Fc=1 Spot=1-r corr
60
M
(oC)
40
30
20
1e-9
1e-8
1e-7
1e-6
1e-5
1e-4
M
Conc
Fc=1 Spot=2-r corr
KD 225nM
@25°C
BRD2_1
50
-20
60
1e-5
120
80
60
40
10
20
0
0
-10
-20
-20
KD <70nM
@25 °C
BRD2_2
100
-40
-50
0
50
100
150
200
250
300
-50
0
50
100
150
200
250
300
 BZD tool binds both N and C-terminal domains
but kinetics and affinity at 25°C are different for each
all Brd2
Isothermal Titration Calorimetry demonstrates
specific binding to both BRDs
1
Brd2 338-473
2
3
 1:1 46nM(16°C)
 1:1 52nM(26°C)
 2:1 30nM(26°C)
Brd2 67-200
Brd2 67-200
Brd2 338-473
ACTIVE BZD
N
N
N
N
O
O
N
N
N
U
U
N
N
ACTIVE BZD
N
N
N
N
N
O
O
O
O
U
U
N
N
GW841819X
N
N
O
O
ACTIVEBZD
NNN
ACTIVEBZD
O NNN
U
N
O
NO
U
N
NO
G W 841 9X
G W 841 9X
GW841819X
N-terminal
bromodomain
C-terminal
bromodomain
I-BET762 is a highly selective inhibitor of BET
bromodomains
Tm profiling
5-7oC
1-3oC
<1oC
I-BET762
iBET Broader Selectivity Profiling
 Inactive against a wide range of proteins
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
N
N
N
GW841819X
O
0.16
ratio
N
N
O
0.12
0.08
0.04
0.0001
0.001
0.01
0.1
[compound] (uM)
1
10
First Small Molecule X-ray co-crystal confirms
binding in the acetylated lysine pocket
GW841819X
N
N
O
U
N
N
O
N
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

Interactions of BZD outside the AcK pocket
N
N
N
O
N
N
O
ACTIVE BZD
BrdT – Nature (2009)
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
Ion Works (Dof)
100uM
Patch Express
61uM
hERG EC50
Rat (Mouse)
PK*
Clb (mL/min/Kg);
Vss (L/kg); t½ (h), %Fpo
Dog PK*
Unbound fraction in blood (R/D/Mou/H)
CYP inhibition IC50s (uM)
P450 TDI
*3mg/kg p.o.; 1mg/kg i.v.
63 (24), 1.8 (1.7), 0.5 (0.8), 27
(22)
5, 1.8, 5.9, 44
0.18 /0.24/0.21/0.19
> 33
<2-fold
Optimisation of dimethyl isoxazole HTS lead to in
vivo probe I-BET 151
HTS Lead
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
<0.53
15
1.6
1.2
65
Human
1.1
BMCL, 2012, 2963
BMCL, 2012, 2968
GSK525762 and GSK1210151 bind BET proteins
using similar “hot spots”
WPF
ZA Channel
I-BET 762
AcK pocket
I-BET 151
Bromodomain Family and Structural Coverage
BRPF1
BRD1
BPRF3
BRD9
BRD7
KIAA1240
ATAD2
CREBBP
EP300
BRD2_1 BRD3_1
BRD3_2
BRD4_1
BRDT_1
BRDT_2
BRD2_2
BRD4_2
BAZ1A
BRD8
WDR9_2
PHIP_2
BRWD3_2
TAF1_1
BAZ1B
PRKCBP1
TAF1L_1
TAF1_2
CECR2 TAF1L_2
FALZ
GCN5L2
PCAF
BAZ2B
BAZ2A
TRIM33
ZMYND11
T
TIF1
TRIM66
Y
SP140
>50 bromodomains
In isolation or
combination with other
domains
T
MLL
TRIM28
Y
SP110
SP100
LOC93349
WDR9_1
T
ASH1L
Y
BRWD3_1
PHIP_1 T
PB1_1
PB1_3
PB1_5 PB1_2
PB1_4
SMARCA4
SMARCA2
Structure known
Atypical AcK Binding Residue
Y
Multiple opportunities
for clinical utility
Across the family there is significant structural
divergence outside of the AcK binding region
BC Loop
ZA Loop
Exploiting Structural Knowledge :
Fragments – Generation of a Hit-ID platform for Bromodomains
N
N
N
N
N
H
O
O
 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
N
N
Me
O
N
O
O
N
O
N
Me
 Confirmation of the binding mode
using crystallography
Me
O
N
O
>20% inhib at 200uM
 Creation of a fragment toolchest that binds in the
AcK recognition pocket of the bromodomain
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
Application of FBBD for Bromodomains
pIC50 BRD 2
pIC50 BRD 3
pIC50 BRD 4
< 4.0
< 4.0
< 4.0 (LE< 0.43)
pIC50 PBMC TNF < 4.7
O
N
WPF shelf
Pharmacophore
O
O
S
N
H
O
N
"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
Application of Encoded Library Technology (ELT)
Structural knowledge
Construction and screening of libraries
L
I
B
R
A
R
I
E
S
1
2
3
TARGETS
Identification of Features
ELT hits against target 2
E
N
R
I
C
H
M
E
N
T
O
N
LIBRARIES
Exploitation of Screening output
1.
Hits
2.
Screening tools
3.
Probes
Preclinical Biology
Nodal AND gene specific intervention?
pI:C
TNFa
IL-6
IFNb
unaffected
blocked
blocked
LPS
I-BET
TNFa
X
X IFNb
IL6
BET compound displaces BRD4 from IFNb and
IL-6 promoters (ChIP)
IFNb
BRD4 / H3
0.25
0.20
IL-6
1.80
DMSO
1.60
BET
1.40
DMSO
BET
1.20
0.15
1.00
0.80
0.10
0.60
0.40
0.05
0.20
0.00
0.00
Control
LPS
Control
LPS
 LPS drives recruitment of Brd4 to selective promoters
 Compounds prevent this recruitment and block transcriptional activation
Soren Beinke
Targeted intervention by I-BET
Primary response genes
Secondary response genes
Pol II
H3K4m3
CpG
high
H3K9Ac
H3K9m1/2/3
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
BET interacting proteins: MS-proteomic analysis
Triple purification strategy
Ac
Nature (2011) 478, 529
Acetylated
H4 tail (K4,K8,K12)
H4
= BET inhibitor
(I-BET)
proteins binding
directly or
indirectly to
histone marks
proteins
binding
directly or
indirectly to
I-BET
BET protein
imunocomplexes
Antibody
against BRD2/3/4
BET interacting proteins: MS-proteomic analysis
3H-thymidine
150
100
100
50
50
0
Cell Viability
150
(% live cells compared
to solvent control)
(% cpm of solvent control)
I-BET762 is effective in multiple models of Multiple
Myeloma
0
-9
-8
-7
-6
-5
-4
**
loglog
(M)
10[GSK525762A]
[I-BET762]
10
Proliferation
Viability
Survival of OPM-2 Xenografts
[I-BET762]
Percent survival
100
80
60
G1 Vehicle qdx58
G2 3mg/kg qdx68
G3 10mg/kg qdx68
40
G4 30mpk(15-31)/20mpk(43-68)
G5 30mg/kg qodx35
20
0
0
20
40
60
80
days after inoculation
100
BET interacting proteins: MS-proteomic analysis
I-BET151 is a novel & selective inhibitor of BET proteins
with improved PK properties
I-BET151
I-BET151 has selectivity for MLL leukaemias
I-BET151 mediates disease control in MLL leukaemia models
NOD-SCID
C57BL/6
Transplant
human MV411
leukaemia cells
Transplant
syngeneic MLL-AF9
leukaemia cells
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
Effect of BET inhibition on LPS induced shock
preventative
therapeutic
I-BET
LPS
I-BET
-1h
0h
1.5h
Nature, 468, p1119, 2010
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)
Kevin Lee