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A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .

High Throughput Kinase Discovery, Inhibitor Screeningand Specificity Analysis using Multi-Array Technology

Nisar Pampori, Pankaj Oberoi, Ryan Swenerton,Ilia Davydov, Stefanie Nelson, Hans A. Biebuyck,

John H. Kenten, and Jacob N. Wohlstadter

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A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .

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AbstractWe developed a high-throughput system for biological discovery and compound screening. A systematic and deterministic approach allowed us to categorize and quantify functional aspects of our library of 22,000 mammalian proteins (based on Unigene) coded by 50,000 individual full-length cDNA clones. Our methodology combined MSD's proprietary Multi-ArrayTM detection technology with high-throughput cell-free production, labeling, immobilization and purification of proteins. Here we report on several approaches we used to identify and characterize proteins with tyrosine kinase activity. First, we screened a set of 20,000 clones (14,000 unique Unigenes) for proteins with tyrosine kinase activity using a screening assay that simultaneously measured auto-phosphorylation and phosphorylation of a consensus tyrosine kinase substrate. The screen recovered known tyrosine kinases, previously unidentified kinases and, under low stringency conditions, proteins that recruited kinases. Second, we selected a redacted set of clones on the basis of homology to known kinases and demonstrated that we could identify ~ 70% of the expected tyrosine kinases in this redacted set. Third, we used MSD Multi-Array technology to test, in parallel, the specificity of 21 of these well-characterized kinases for a panel of known kinase inhibitors. Finally, we carried out a focused screen of 2,000 compounds for inhibitors of two of the identified kinases. The combination of high-throughput protein expression and Multi-Array technology allows for a unified approach to the discovery of biological targets and the identification of small molecules that regulate their activity.

High Throughput Kinase Discovery, Inhibitor Screeningand Specificity Analysis using Multi-Array Technology

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A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .

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High Throughput In-Vitro Expression Screening for Protein Tyrosine Kinase Activity

Multi-Array Plate

α−PhosphotyrosineAntibody (α−pY)

α−PhosphotyrosineAntibody (α−pY)

Substrate

In-situ protein expression

Expressed protein

Capture protein

Multi-Array Label (MSD TAGTM)

Biotinylated α−pY

Streptavidin

Multi-Array Label(MSD TAGTM)

Kinase activity assay

Kinase activity was determined by capturing the phosphorylated

substrate with an anti-phosphotyrosine (α-pY) antibody and detection using

MSD Multi-Array technology.

Autophosphorylation Assay

The extent of auto-phosphorylation of the immobilized tyrosine kinase was detected using MSD Multi-Array

technology.

cDNA clones

Clones expressed and labeled in 96-well format

Expressed protein immobilized on plate and purified

Kinase phosphorylates consensus substrate or itself (autophosphorylation)

Transfer to second plate for assay

Autophosphorylation(assay in same plate)

ATP

ATP

Phosphate

High Throughput Kinase Discovery, Inhibitor Screeningand Specificity Analysis using Multi-Array Technology

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A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .

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Screening of 20,000 cDNA Clones

Results

Evenly Spaced Controls through 1 week ofscreening (10,000 clones)

Validation

50,000 cDNA clones

Immobilized on plate

Tyrosine Kinase assay on Sector HTSTM Reader

Identified Kinases and Kinase binding protein

Entire Process <1 month

20,000 clones expressed and labeled in a 96-well format.

1000Batch 1 Batch 2 Batch 3 Batch 4 Batch 5 Batch 6

10000

100000

1000000

10000000

SYK BTK FERT2 Neg. ControlsKinases

20,000 Clones

Stand

ardize

d Sig

nal

S YK

MATKSCAP2

HCKFGFR1

LynBTK

FGRFRK

HCK

25 50 75 100 125 150 175 200 225 250 275 300

0

100

200

300

400

Kinase BindingProtein (SCAP2)

Known Tyrosine KinasesOther Hits

High Throughput Kinase Discovery, Inhibitor Screeningand Specificity Analysis using Multi-Array Technology

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A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .

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Kinase Activity from Our LibraryDemonstration of tyrosine kinase specificity of our assay across the kinase genome

0

100

200

300

400 300 200 100 0

GenomeMSD LibraryHits

Tyrosine Kinase Homolog

Serin

e-Threonin

e Kin

ase H

omolo

g

Ser/Thr KinaseHomology

Tyr KinaseHomology

Kinaseactivity

Positive kinases areshown in yellow.

Kinases in the genome and kinases in our library bygene similarity score. The overlap in the two distributionsdemonstrates the high degree of coverage of our library.Putative kinases scored as having high homology to known tyrosine kinases and confirmed as such in our activity-based assays.

High Throughput Kinase Discovery, Inhibitor Screeningand Specificity Analysis using Multi-Array Technology

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A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .

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Tyrosine Kinase Family Tree

Protein Tyrosine Kinases Active fromHigh Throughput Expressed Clones

EPHA3EPHA5

JAK2

JAK1

SYKZAP70

JAK3TYK2

EPHA1INSR

INSRR

IGF1R

LMR2LMR1

TNK1ACK1

PTK2PTK2B ERBB4

ERBB2ERBB3

DDR1DDR2

ROS1

RYKMST1R

NTRK1

MUSKPTK7

ALKLTK

RET

FGFR4

FGFR3

FGFR2 FGFR1

TEK

FLT3

FESFER

BMX

ABL1ABL2

YES1SRC

FGR FYNLCK

FRKCSK

MATK

BLK

HCK LYN

TXKBTK

TECITK

FLT1KDRFLT4 KIT

CSF1R

PDGFRA

TIE

ROR1/2

NTRK2NTRK3

AXLMERTK

MET

TYR03

EGFR

EPHB2EPHB1

EPHB3

EPHB4

EPHB6 EPHA2

EPHA4EPHA7

EPHA8

- Kinases from Screens

- Kinases used in specificity study

KIN

PDGFRB

Consensus substrate Autophosphorylation

100

1,000

10,00

0

100,0

00

1,000

,000

1,000

10,00

0

100,0

00

1,000

,000

10,00

0,000

Signal on Sector HTS

hemopoieticcell kinaseGardner-Rasheedfeline sarcoma viral (v-fgr) oncogenehomolog

protein tyrosine kinase2 betac-src tyrosine kinase

Eph receptor A7fibroblast growth factor receptor 1fer (fms/fps related) protein kinase

Bruton agammaglobulinemia tyrosine kinaseYamaguchi sarcoma viral (v-yes-1) oncogenehomolog

FYN oncogenerelated to SRCendothelial-specific receptor tyrosine kinase

kit oncogeneRoussarcoma oncogene

kinaseinsert domain protein receptorEph receptor B4

fibroblast growth factor receptor 4megakaryocyte-associated tyrosine kinase

B lymphoid tyrosine kinasespleen tyrosine kinase

neurotrophic tyrosine kinaseB-cell src-homology tyrosine kinasefibroblast growth factor receptor 2

fyn-related kinaselymphocyte-protein tyrosine kinase

CDC-like kinase2Yamaguchi sarcoma viral (v-yes) oncogenehomolog

BMX non-receptor tyrosine kinaseJanuskinase 1 (a protein tyrosine kinase)

EphB1Eph receptor B3

anaplastic lymphoma kinase (Ki-1)hemopoieticcell kinase

FYN oncogenerelated to SRCCDC-Like Kinase1CDC-like kinase3

fibroblast growth factor receptor 1 (fms-related tyrosine kinase 2)CDC like kinase 4TTK protein kinase

wee 1 homolog (S. pombe)lymphocyte-specific protein tyrosine kinase

controlvector

Negatives(n=10)TK substrate

TK binding protein

High Throughput Kinase Discovery, Inhibitor Screeningand Specificity Analysis using Multi-Array Technology

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A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .

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Multi-Array Tyrosine Kinase Specificity of Inhibitors Towards In-Vitro Tyrosine Kinase Activity (Autophosphorylation and Substrate)

Hierarchical cluster analysis of inhibitors and kinases

A survey of known inhibitors reveals patterns of their actions on a set of tyrosine kinases, allowing classification of these kinases into groups reflecting their lineage. The individual drug profiles are useful supplements to general studies of the physical organic basis of chemical phenotypes.

Tyrosine KinasesAutophosphorylation Consensus substrate

Compound Damnacanthal

AG-1433

VEGFR2 Inhibitor

WHI-P131

VEGFR Inhibitor

PP1

Oxindole 1

AG-1024

AG-957

LFM-A13

Erbstatin analog

Lavendustin A

AG879

AG-17

Genistein

AG-1296

AG-1478

PP2

Compound-56

SU-1498

AG-490

AG-538

LCK

PDGFR

KDR (Vegfr2)

Jak3

KDR (Vegfr2)

FYN

Flk1

IGF1R

Abl

BTK

EGFR

EGFR

NTRK1

PDGFR

EGFR

PDGFR

EGFR

LCK

EGFR

Flk1

EGFR

IGF1R

0.62

5

0.07

7.8

2

0.6

0.39

18

0.75

17.2

0.78

0.011

10

0.5

2.6

1

0.003

0.004

6-pM

0.7

0.1

0.06

Known EffectIC50(µM)

BTK Kit Kdr

FGFR

1FG

FR2

BMX Src

Fert2

Fgfr-

4CSK

SYK

HCK

FRK

PTK2

BEp

hb4

Tnk1

NTRK

3Ep

ha7 Tek FGR

LCK

BTK Kit Kdr

FGFR

1FG

FR2

BMX Src

Fert2

Fgfr-

4CSK

SYK

HCK

FRK

PTK2

BEp

hb4

Tnk1

NTRK

3Ep

ha7 Tek FGR

LCK

Relative Kinase Activity0 1 2

(10X IC50)

High Throughput Kinase Discovery, Inhibitor Screeningand Specificity Analysis using Multi-Array Technology

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A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .

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From Biological Discovery to Compound DiscoveryRapid progression to titratable inhibitors for chemical genomic and other types of study. The biological screening format used previously is also employed here to screen compounds.

50,000 cDNA clones

Express and label

Immobilized on plate

Screen Kinase for Inhibitor

Identified Kinases

0

20

40

60

80

100

120

140

Sample Number

Z > 0.5

0 20 40 60 80 100 120 140 160

0

20

40

60

80

100

120

140

160

0.01 0.1 1 10 1000

25

50

75

100

125A

A

% Acti

vity

% Acti

vity

Autop

hospho

rylati

on (H

CK)

% Acti

vity

Autop

hospho

rylati

on (H

CK)

% ActivityAutophosphorylation (FGFR1)

Concentration (µM)

IC50~ 600nM

High Throughput Kinase Discovery, Inhibitor Screeningand Specificity Analysis using Multi-Array Technology

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A d i v i s i o n o f M e s o S c a l e D i a g n o s t i c s , L L C .

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SummaryThe combination of Multi-Array Technology with our unique cDNA library and high throughput methods for protein expression and labeling powerfully augments existing paradigms of drug discovery and development.We showed the application of these techniques to basic discovery, inhibitor screening and specificity analysis of tyrosine kinases and their substrates, demonstrating an effective and available “clone to screen” strategy. We validated this solution in real time, taking an identified clone through a 2,000 focused compound library screen in less than a day.

We offer:•Discovery solutions on a genome scale for kinases and other activities in cDNA libraries and clone collections. We have genome scale screens for proteins involved in ubiquitinylation, phosphorylation, protein binding, phosphorylated protein binding, and kinase substrate determination.•Compound specificity analysis for inhibitors in high throughput formats that allow practical profiling and categorization of thousands of lead compounds against panels of targets.•Clone to screen solutions.•Custom screening of the MSD clone library comprising 22,000 unique genes among 50,000 members (based on the Unigene classification), largely full length and sequence confirmed. Demonstrated formats include invitro assays for ubiquitinylation, phosphorylation, protein:protein interactions, phosphorylated protein:protein interactions, kinase substrates discovery, acetylation, proteolysis, and post-translational modification. •Cell-based assays.

Meso Scale Discovery, MSD, MSD (design), Multi-Array, Multi-Spot, and Sector HTS are trademarks of Meso Scale Diagnostics, LLC.© Meso Scale Discovery, a division of Meso Scale Diagnostics, LLC. All rights reserved.

High Throughput Kinase Discovery, Inhibitor Screeningand Specificity Analysis using Multi-Array Technology