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“ Bromodomains : A new class of epigenetic targets ripe for small molecule drug discovery” . Jason Witherington EpiNova DPU. ELRIG – Manchester 2012. Outline. Brief introduction to Epigenetics Luck strikes!.....discovery of small molecule bromodomain inhibitors - PowerPoint PPT Presentation
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“Bromodomains: A new class of epigenetic targets ripe for small molecule drug discovery”
Jason WitheringtonEpiNova DPU
ELRIG – Manchester 2012
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
· 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
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
NN
R2N
R1
N
Benzodiazepine
5’-UTR ApoA1
3’-UTR ApoA1
-1.4kb
Human ApoA1 promoter
Firefly luciferase
How were new medicines discovered ?
Nat. Rev Drug Discovery 10, 507 (2011)
Between 1998-2008:
More first-in-class drugs were discovered by phenotypic screening
More follower drugs were discovered by target-based screening
1999-2008
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
PROTEINIDENTITY
Biologically relevantsystem
HepG2 & THP1
Derivatised CompoundsActive BZDInactive BZD
NN
R2N
R1
N
NN
R2N
R1
N
J Med Chem (2011) 54, 3827
200
4050
150kDa
3020
10075
BZD
act
ive
mat
rixB
ZD In
activ
e m
atrix
Mat
rix a
lone
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
+Ser
ies
X in
activ
e
200
40
50
150
kDa
3020
10075
+ B
ZD a
ctiv
e
+ B
ZD in
activ
e
+ Se
ries
X ac
tive
All bands identified as BET family proteins Brd2, Brd3, Brd4
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 – 1mM BRD4 siRNA: 500nM
DC
T
24hr
48hr 96hr
0hr
24hr 48hr
72hr
-1.6
-1.4
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
0.2
1
Incr
ease
in A
po-A
1 m
RN
A
BET binding correlates with Apo-A1 cellular activity
BZDs
BRD4 FP p IC50
Apo
-A1
pec1
70
NO
NN
N
N
O
Cl
I-BET762
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
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 domainbromodomain2bromodomain1
Ac Ac Ac
Acetylated lysines on Histones within euchromatin
Pol II
transcriptionBRD BRD
pTef-B
P
ApoA1 compounds bind to BET BUT where specifically do the compounds interact?
Chemoproteomics implicate Bromodomain of Brd2, 3, 4
Bromodomain 1 Bromodomain 2 ET domain
X
22012010080
60
4050
30
20
kDa
1. GFP control
2. Flag Brd2 FL
3. Flag Brd2 N
4. Flag Brd2 C
Hek293 cells
1 2 3 4PD: BZD active
Western Blot: anti FLAG
1 801
473
FL
N
C
X
Biophysical data demonstrates specific binding
Some BIAcore Results - GW842819X BZD active
-20
0
20
40
60
80
100
120
1e-9 1e-8 1e-7 1e-6 1e-5 1e-4
Resp
on
se
RU
Conc M
Steady State: Fc=1 Spot=2-r corr
-10
0
10
20
30
40
50
60
1e-9 1e-8 1e-7 1e-6 1e-5
Res
po
nse
RU
Conc M
Steady State: Fc=1 Spot=1-r corr
-20
-10
0
10
20
30
40
50
60
-50 0 50 100 150 200 250 300
Fc=1 Spot=1-r corr
-40
-20
0
20
40
60
80
100
120
-50 0 50 100 150 200 250 300
Fc=1 Spot=2-r corr
Brd2 67-200 Brd2 338-473
KD = 2.25e-7MRmax = 41 => 40% surfaceis active.
KD < 7.0e-8MRmax = 74 => 70% surfaceis active.koff ~ 0.01s-1 ± 0.03 s-1
kon
N
NN
N
N O
O
U
GW841819X
KD 225nM@25°CBRD2_1
KD <70nM@25 °CBRD2_2
BZD tool binds both N and C-terminal domains but kinetics and affinity at 25°C are different for each
40 50 60
norm
alis
ed C
D
• 0
0.2
0.4
0.6
0.8
1 Brd2(1-473)
Temp (oC)
INACTIVE X
INACTIVE Y
ACTIVE X
ACTIVE Y
Tool compounds stabilise all Brd2 bromodomain constructs
ACTIVE X INACTIVE X
ACTIVE Y INACTIVE Y
Isothermal Titration Calorimetry demonstrates specific binding to both BRDs
C-terminalbromodomain
N-terminalbromodomain
N
NN
N
N O
O
U
GW841819X
ACTIVE BZD
N
NN
N
N O
O
U
GW841819X
ACTIVE BZD
Brd2 67-2001
Brd2 67-200 Brd2 338-4733
Brd2 338-4732
C-terminalbromodomain
N-terminalbromodomain
N
N
N
N
NO
O
U
GW841819X
ACTIVE BZD
N
NN
N
NO
OU
GW841819X
ACTIVE BZD
1:1 46nM(16°C)
1:1 52nM(26°C)
2:1 30nM(26°C)
N
NN
N
N O
O
U
GW841819X
ACTIVE BZD
N
NN
N
N O
O
U
GW841819X
ACTIVE BZD
I-BET762 is a highly selective inhibitor of BET bromodomains
Tm profiling
5-7oC1-3oC<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
[compound] (uM)0.0001 0.001 0.01 0.1 1 10
ratio
0.04
0.08
0.12
0.16 GW841819X
FRET assay for displacement of tetraacetylated H4
N
NN
N
N O
O
N
NN
N
N O
O
U
GW841819X
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
Interactions of BZD outside the AcK pocket
N
NN
N
N O
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
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.
Optimisation of dimethyl isoxazole HTS lead to in vivo probe I-BET 151
N
O
O
N
NH
N
O
N
NO
N
NH
NH2
O
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, 2963BMCL, 2012, 2968
HTS Lead I-BET 151
GSK525762 and GSK1210151 bind BET proteins using similar “hot spots”
WPF ZA Channel
AcK pocket
I-BET 762
I-BET 151
NN
NN
HN
O
O
Cl
N
O
ON
HN
N
ON
Bromodomain Family and Structural CoverageBRPF1
BRD1BPRF3
BRD9BRD7
KIAA1240ATAD2
WDR9_2PHIP_2
BRWD3_2
BAZ2B
BAZ2A ZMYND11
CREBBPEP300
BAZ1A
BRD8
BRDT_1
BRD4_1
BRD2_1 BRD3_1BRD3_2
BRDT_2
BRD2_2
BRD4_2
TAF1_1
TAF1L_1TAF1_2
TAF1L_2
PRKCBP1
CECR2FALZ
GCN5L2PCAF
WDR9_1
BRWD3_1PHIP_1
ASH1L
PB1_1PB1_3
PB1_2PB1_4
PB1_5
SMARCA2SMARCA4
TRIM33
TIF1TRIM66
MLL
SP110
SP100LOC93349
SP140
TRIM28
BAZ1B
Structure knownAtypical AcK Binding Residue
T
T
T
T
Y
Y
Y
Y
>50 bromodomainsIn isolation or combination with other domains
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
Knowledge of key ligand-protein interactions derived from the Bet programme lead-like compounds
NN
N
N
NH O
O
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
N
O
N
O
Confirmation of the binding mode using crystallography
>20% inhib at 200uM
NMe O
N
OMe
O NN
OMe
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 identified Pharmacophore refined
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
O
N
S NH
O
O
pIC50 BRD 2 pIC50 BRD 3 pIC50 BRD 4
5.2 5.9 5.6 (LE 0.38)
pIC50 PBMC TNF 6.5
PharmacophoreWPF 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-waACCEPTED
Application of Encoded Library Technology (ELT)
NO
LIBRARIES
ENRICHMENT
LIBRARIES
1 2 3 TARGETS
Structural knowledge Construction and screening of libraries
Identification of Features
ELT hits against target 2
Exploitation of Screening output
1. Hits
2. Screening tools
3. Probes
Preclinical Biology
Nodal AND gene specific intervention?
X X
pI:C LPS
TNFaIL6 IFNb
I-BET
unaffected blocked blocked
TNFa IL-6 IFNb
BET compound displaces BRD4 from IFNb and IL-6 promoters (ChIP)
LPS drives recruitment of Brd4 to selective promoters
Compounds prevent this recruitment and block transcriptional activation
BR
D4
/ H3
IL-6IFNb
Control LPS0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40DMSOBET
Control LPS0.00
0.05
0.10
0.15
0.20
0.25DMSOBET
Soren Beinke
Targeted intervention by I-BET
Secondary response genesPrimary response genes
CpG high
H3K4m3
H3K9Ac
Pol IIH3K9m1/2/3
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
proteins binding
directly or indirectly to
I-BET
proteins binding directly or
indirectly to histone marks
BET protein imuno-
complexes
= BET inhibitor (I-BET)
AcAcetylated
H4 tail (K4,K8,K12)
H4
Antibody against BRD2/3/4
Triple purification strategy
BET interacting proteins: MS-proteomic analysis
Nature (2011) 478, 529
BET interacting proteins: MS-proteomic analysis
log10[GSK525762A] (M)
3H-t
hym
idin
e(%
cpm
of s
olve
nt c
ontr
ol)
Cell Viability
(% live cells com
paredto solvent control)
-9 -8 -7 -6 -5 -40
50
100
150
0
50
100
150
ProliferationViability
I-BET762 is effective in multiple models of Multiple Myeloma
**
[I-BET762] Survival of OPM-2 Xenografts
0 20 40 60 80 1000
20
40
60
80
100
G1 Vehicle qdx58
G2 3mg/kg qdx68
G3 10mg/kg qdx68
G4 30mpk(15-31)/20mpk(43-68)
G5 30mg/kg qodx35
days after inoculation
Perc
ent s
urvi
val
log10 [I-BET762]
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
Transplant human MV411 leukaemia cells
Transplantsyngeneic MLL-AF9 leukaemia cells
C57BL/6
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
0h
LPS
therapeutic
I-BET
1.5hpreventative
I-BET
-1h
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
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