BROMOscan Quantitative Ligand Binding Assays

Identification

of

Best

‐

In

‐

Class

Bromodomain

Inhibitors

Elizabeth

Quinn,

PhD

Director,

LeadHunter

Discovery

Services

Outline

•

Introduction

•

Challenges

to

Epigenetic

Drug

Discovery

•

Solution

 ‐

BROMO

scan

Technology

Platform

What

is

Epigenetics?

Arrowsmith, et al.  Nature Rev Drug Disc.11: 384‐400, 2012.

• Our DNA is impacted by several environmental & 

genetic factors

• Addition of methyl groups and histone tail modifications

• Combination of ‘marks’ determine gene expression 

• Leads to health issues ‐ cancer, diabetes, autoimmunity

Gene

regulation

independent

of

DNA

sequence

–

determines

whether

genes

are

turned

on

or

off

Histone

Code

•

Histones

– globular

proteins

wrapped

in

DNA

•

Histone

tails

– subject

to

a

wide

range

of

covalent

interactions

•

Epigenetic

marks

– addition

&

removal

of

chemical

groups

•

Acetylation

of

lysine

residues

(Kac)

‐

most

frequently

occurring

post

‐

Epigenetic

Proteins

Modification Write Erase Read

Acetyl HAT HDAC Bromo

Methyl HMT HDM Chromo, PHD, Tudor, MBT

Enzyme Enzyme Binding

A Maxmen, Nature 

GSK 525762

OTX105 RVX‐208

•

Evidence

for

small

molecule

inhibition

of

readers,

writers,

erasers

•

Mainly

HDAC

inhibitors

in

clinic

until

recently

•

Diazapines

are

selective

BET

inhibitors

Oncology  Inflammation 

DeWoskin and Million, NDD, 12: 661‐662, 2013.

Therapeutic

Potential

of

Bromodomains

•

Basic

understanding

of

transcription

•

Novel

approaches

to

cancer

biology

– AML,

NUT

midline

carcinoma,

myeloma,

glioblastoma

Challenges

for

Screening

Bromodomains

Chemical

Probes/drugs

•

Assay

development

difficult

•

Available

technologies

have

drawbacks

•

Difficult

to

run

as

a

large

assay

panel

Need

for

a

large

menu

of

assays,

HTP

screening

tool

Assay Output Sensitivity Throughput Ligand Needed? Limitation

AlphaScreen Low IC50 High High Yes High false positive rate

Tm Shift High °C Medium High No Indirect

Octet BLI Low Kd Medium Medium No Biotinylated protein

KINOME

scan®

Technology

‐

The

Solution

KINOMEscan

applied to BRDs

World’s

Largest

Kinase

Panel

• Cover >80% human kinome

• 456 assays – lipid, TK, mutants

• Enables new drug discovery paradigms

Our

Goal

for

Bromodomains

• Leverage our expertise 

• Build a first in class panel of 

BROMO

scan

Technology:

How

it

Works

Measure amount of bromodomain bound 

to immobilized ligand in the presence and 

absence of test compound Competition No Competition

‐ Test Compound + Test Compound

Ultrasensitive qPCR readout (6‐log range)

BROMO

scan

Menu

BRD1 BRD4(full‐length, short‐iso) CECR2 TAF1(2)

BRD2(1) BRD7 CREBBP TAF1L(2)

BRD2(2) BRD8(1) EP300 TRIM24(Bromo.)

BRD2(1,2) BRD8(2) FALZ TRIM24(PHD,Bromo.)

BRD3(1) BRD9 GCN5L2 TRIM33(PHD,Bromo.)

BRD3(2) BRDT(1) PBRM1(2) WDR9(2)

Roman numerals indicate SGC‐defined families

Human Bromodomain 

Dendrogram

40 Human Bromodomain Assays  

• 34 of 57 distinct bromodomains (~60% coverage) 

• Multiple putative therapeutic targets: BET, ATAD2, 

CREBBP/EP300, TRIM24(PHD,Bromo.)

• Ability to perform K_d FUP studies on single vs 

tandem domains

*    BRD2, BRD3, BRDT tandem assays also available

BROMO

scan

Assay

Validation

•

Small

Molecules

‐

Measure

K

s

consistent

with

known

ITC

values

(when available)

‐

Inhibitors:

•

JQ1

(active

enantiomer)

•

I

‐

BET

(active

and

inactive

enantiomers)

•

PFI

‐

1

&

Bromosporine

(SGC)

and

other

known

BRD

inhibitors

•

Acetylated

Histone

Tail

Peptide

Ligands

‐

Measure

K

s

consistent

with

published

values

(when available)

‐

Demonstrate

selectivity

for

acetylated

over

non

‐

Bromosporine Kd(nM)

Tm

Shift (100

uM Bromosporine)

Comparison

of

BROMO

scan

and

SGC

T

Shift

Data

for

the

Promiscuous

Inhibitor

Bromosporine

• 24/25 BROMOscanassays analyzed

• Each square represents a unique bromodomain 

• Outstanding agreement between BROMOscan

and TmShift data

− Cross‐validates the formats

− Further quantifies Bromosporine promiscuity

Striking

agreement

between

two

very

different

assay

readouts

Global

Validation

of

BROMO

scan

Panel

SGC T_mshift data provided by S. Knapp, PhD 

Assay

Validation:

Family

II

(BET)

Family

II

(BET)

assays

•

BRD2(1)

•

BRD2(2)

•

BRD3(1)

•

BRD3(2)

•

BRD4(1)

•

BRD4(2)

•

BRDT(1)

•

BRDT(2)

BET

Family

Assay

Validation

–

JQ1

Kds

Correlate

with

Literature

Values

*Nature(2010) 408: 1067

Data

Comparison

• Similar Kd values measured with BROMOscan & ITC

‐ Domain affinity rank order identical

• Optimized conditions ensure true thermodynamic 

BET

Family

Validation

I

‐

BET

Nature(2010) 468: 1119.

BROMOscan data consistent with published ITC data

• Potency

• Rank order

Selective Binding to Acetylated Histone Tail Peptide

1_{internally discovered acetylated peptide ligand} 2_{corresponding non‐acetylated peptide}

Ligand Affinity Measurements

Also determine K_dValues of 1‐50 uM for 

other known BRD  inhibitors

Bromodomain Histone Tail  Peptide BROMOscan  Kd (nM) BRD1 H4(Ac)1 15,000 BRD1 H42 >100,000

Assa

y

Signal

[Ligand],

nM

Family

IV

Validation:

BRD1

Bromodomain

Inhibitor

Profiles

I

‐

BET

Bromosporine

BET

‐

Selective

Promiscuous

PFI

‐

1

Dual

Kinase/Bromodomain

Inhibitors

 ‐

Rationally

Designed

Polypharmacology

•

Question:

–

Do

kinase

inhibitors

cross

‐

react

with

bromodomain

epigenetic

reader

proteins?

•

Experiment:

–

Screen

diverse

panel

of

mature

kinase

inhibitors

across

internal

bromodomain

assay

panel

(BROMO

scan

)

•

Implications:

–

Outstanding

leads

for

BRD

inhibitor

discovery

–

Proof

of

principle

for

development

of

dual

kinase/BRD

inhibitors

Result:

– Potent (Kd = 0.01‐ 1 M), diverse BRD profiles for 4/68 (5.9%) kinase inhibitors tested

BROMO

scan

Bromodomain

Potency

and

Selectivity

BROMO

scan

Summary

Specific

Sensitive

Quantitative

HTS

Compatible

Generic

Assay

• Accurate Kd values over a broad dynamic range 

<100 pM to >10 uM 

• Optimized conditions for true thermodynamic 

affinity (Kd) measurements

• Detect small amts of protein, low amts of kinase

• Screen more than 100,000 wells/day

• No interference from compound fluorescence • Unique, semi‐automated set‐up

• Generic, standardized assay conditions • Industrial scale screening & profiling

• Fast time to results ‐2 week data delivery

• Rigorous assay validation with optimized bait ligands • Heterogeneous– screen to solubility limit of compound • No fluorescent compound interference

Flexible

Service

Menu

Service Number of 

Assays

Description

32 Growing panel of 32 human bromodomains 

validated for single point testing

1-32 Choose only the targets you need for 

personalized or custom panels

1-40 Quantitative binding constants on any of the 40 

bromodomain assays

40 Kd profile of entire BROMOscan menu

1-32 Compound library screening against a single 

target or against the full panel

DiscoveRx

Solution

Portfolio

>80%

Coverage

of

Druggable

Targets

+

Primary

Cell

Phenotypic

Assays

HDAC/HMT Depth   of   Cov e ra ge 100% 0% 284 394 21 23 40

GPCRs Kinases NHRs Pathways Bromodomain

1,120 assays covering 751 druggable targets

• All major drug target classes covered

Acknowledgements

•

Dan

Treiber

•

Jeremy

Hunt

•

Lisa

Wodicka

•

Pietro

Ciceri

•

Lisa

Ramos

•

Mark

Floyd

•

Gabe

Pallares

•

KINOME

scan

Team

•

Jay

Bradner

•

Jun

Qi

•

Stefan Knapp