[PDF] - Overview Brief introduction to epigenetics and bromodomains PDF Document

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Targeting Bromodomains – BET and the wider target class

DiscoveRx & SGC Symposium - 12th September 2013 Matthew Lindon

Overview

Brief introduction to epigenetics and bromodomains Identification of BET inhibitors Delivering selectivity in the BET family Beyond BET and target class approaches to other Bromodomain family

members

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Chromatin & Epigenetics

DNA is packaged around histones and

ther 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) post translational modifications (PTMs)

n histone tails

Dysregulation of histone PTMs implicated in human disease

Histones: Post-translational modifications

PTM of histone tails

PTMs can have a direct impact on physical properties
f individual nucleosomes e.g. neutralisation of charge
PTM are recognised by specialised reader domains.

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Bromodomains bind to acetylated lysine residues

N N N H O O N N

Lysate from HepG2 Princen THP1 or HEK293 5-10 x 108 cells per pulldown Modified Compound Jonathan Wilde Helen Flynn Julia White

Serendipity - BET family BRDs as tractable targets

“Black Box” transcriptional screen identified a series of ApoA1 up-regulators (CVU)

Matrix

p attached to Reactigel Separate

n 1-D

Gel LC/MS/MS MS: DBP PROTEIN IDENTITY Data Analysis Elution with SDS A I PMM DDA

GW841819X Series

Some BIAcore Results - GW842819X BZD active

RU Steady State: Fc=1 Spot=2-r corr RU Steady State: Fc=1 Spot=1-r corr

Brd2 67-200 Brd2 338-473

N N N O U

20

20 40 60 80 100 120 1e-9 1e-8 1e-7 1e-6 1e-5 1e-4 RU Co nc M Steady State: Fc 1 Spot 2 r corr

10

10 20 30 40 50 60 1e-9 1e-8 1e-7 1e-6 1e-5 RU Conc M Steady State: Fc=1 Spot=1-r corr

20
10

10 20 30 40 50 60

50

50 100 150 200 250 300 Fc=1 Spot=1-r corr

40
20

20 40 60 80 100 120

50

50 100 150 200 250 300 Fc=1 Spot=2-r corr

KD = 2.25e-7M Rmax = 41 => 40% surface is active. KD < 7.0e-8M Rmax = 74 => 70% surface is active. koff ~ 0.01s-1 ± 0.03 s-1 kon

N N O U GW841819X

Target confirmed as the BET family of bromodomains

Chung et al - Journal of Medicinal Chemistry (2011), 54(11), 3827-3838

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BET Bromodomain Inhibition

BET family made up of Brd2, Brd3, Brd4 and BrdT Each family member has two distinct bromodomain

binding pockets

Inhibitors published to date typically bind all 8 of

the BET bromodomains with similar affinity, somewhat limiting their utility as specific probes

Growing body of literature demonstrates Pan-BET

inhibition leads to profound preclinical biology

Is it possible to identify selectivity within the

BET family?

Brd4 BD1 Brd4 BD2

BET Selectivity – Reason to believe (I-BET151)

Very high domain

Brd4 BD2 Asp144 His437

sequence homology would suggest selectivity will be difficult to achieve

Key residues for

targeting identified

Chemistry focused on a

specific vector

Tyr97 Tyr390

How will we know we’ve been successful?

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Brd4 Bromodomain Mutants

Requirement for robust reagents and assays Brd4 protein mutations

Brd4 Y390A Mutation of C terminal BD2 Bromodomain

–

Brd4 Y390A – Mutation of C-terminal BD2 Bromodomain

–

Brd4 Y97A – Mutation of N-terminal BD1 Bromodomain

–

Brd4 Y97A Y390A – Mutation of both Bromodomains

Y390A N C BD1 BD2 N C Wild type Y97A N C Y390A N C Y97A Y390A N C

Ligand Based Assays

360 380 400

Brd4 Wild Type Brd4 Y97A

1e-1 1 1e1 1e2 1e3 mP 180 200 220 240 260 280 300 320 340 360 [Brd] (M) Kd (nM) Ser (nM) Brd4 Wild type 11.2 0.6 Brd4 Y97A 18.2 1.1 Brd4 Y390A 19.2 1.4 Brd4 Double Mut > 1000

Brd4 Y390A Brd4 Double Mutant

Titrations of Brd4, Brd4 Y97A, Brd4 Y390A and Brd4 Y97A

Y390A against 5nM AF647 benzodiazepine ligand

n=6 (replicates)

[Brd4] (nM)

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I-BET151 to BD1 Selective I-BET

%I

Conc.

200-fold domain selectivity demonstrated within this series Probe quality and utility is highly dependent on selectivity

Brd4 BD1 Brd4 BD2 IC50 nM (n) 32 (4) 6300 (4)

Selective I-BET

Platform approach to screening assays has enabled a wide range of

profiles to be identified and optimised

DiscoveRx BROMOscan panel confirms target class wide selectivity

Increasing BET selectivity

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Wider Bromodomain Target Class

>50 bromodomains In isolation or combination

with other domains

Multiple “untapped”

pportunities for clinical

utility

Rapid entry into Probes to

confirm phenotype

Structural divergence outside of the AcK pocket

BC Loop ZA Loop

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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 compounds

Generation of a pharmacophore model Selection of a focused screening set Creation of bespoke fragment set that binds in the AcK recognition pocket of the bromodomain Confirmation

f

the binding mode using crystallography

N O

N O N N H O

Extending Fragment Based Discovery

1400 Fragments screened >40 Fragments crystallised Key Structural waters identified Pharmacophore refined

Chung et al - Journal of Medicinal Chemistry (2012), 55(2), 576-586

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Utilising Structural Knowledge to Develop Assays

Small molecule inhibitor crystallised with PCAF

bromodomain

Derivative tagged with AlexaFluor488

FP ligand binding shown to be competed with

acetylated peptides, acetyl lysine

Kd for PCAF BRD mutant ~ 100-fold higher than for

WT

240 260 280 [PCAF] (uM) 0.01 0.1 1 10 100 mP 120 140 160 180 200 220 240 PCAF Brd Mutant PCAF Wild Type

Expansion into Bromodomains with close homology

320 340 360 380

PCAF BPTF

[Novel Brd] (uM) 1e-4 1e-3 1e-2 1e-1 1 1e1 mP 160 180 200 220 240 260 280 300 320

BPTF ZMY11 CECR2 GCN5 ATAD2A

FP tagged ligand profiled against Bromodomain containing proteins with close

domain homology

Additional utility demonstrated for GCN5, FALZ (BPTF) and CECR2

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Development & Utility of a Ligand Based Library

Utilised GSK’s Bromodomain chemical and structural knowledge to build an

extensive library of small molecule ligands

Profiled ligands against broad panel of Bromodomain containing proteins

Hits for multiple targets; cross reactivity gives broad target class coverage Screening assays developed and novel hits identified Probe discovery platform capability established Strategic collaboration initiated with DiscoveRx Inc.

Target S l ti it Target

Epinova Chemical Probe Strategy

Target Selection & Probe ID

Selectivity Engagement Target Phenotype Target Effect

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Summary

Phenotypic screening led to the identification of a novel druggable

epigenetic target class

Further refinements have delivered potent, selective BET inhibitors
Identifying novel areas of biology for drug discovery programmes

requires the identification of high quality chemical probes T t l k l d h bl d th t bli h t f hi h

Target class knowledge has enabled the establishment of a high

quality Bromodomain inhibitor identification platform

Breadth of knowledge and approaches has enabled rapid and

efficient identification of novel potent and selective probes across the bromodomain family

Acknowledgments

Rab Prinjha & colleagues within the Epinova DPU Stuart Baddeley, Chun-wa Chung, Inmaculada Rioja, Chris Wellaway, Peter

Craggs, Ryan Bingham, Laurie Gordon, Natalie Barrett, Paul Bamborough, Lee Harrison, David Hirst and Hannah Traynor

Many colleagues within GSK across MDR Chemical and Biological Sciences DiscoveRx Inc – Paul Gallant, Daniel Treiber

Kevin Lee

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