Applications of KREX for Kinases€¦ · A BP c o ne tr a i ( u M) R F U CDK2 [B] 0 25 75 100 125 150 5000 10000 15000 A BP c o ne tr a i ( u M) R SR C [B ] 0 25 50 75 100 125 150

Applications of KREX for Kinases

© 2008 - 2019© 2008-2019 Sengenics Corporation Pte Ltd - Singapore

we measure autoantibodies

at the highest sensitivity and reproducibility

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Ultricies Ullamcorper


Our background and history3

Prof Jonathan BlackburnInventor and CSO

Fundamental KREX technology patented

and developed

Globally commercialized in 2016

USA, EU and East Asia

Acquired KREX outright Developed industrial version

New apps


Customers and collaborators4

O S L O C A N C E R C L U S T E R


The core principle of KREX™5

▪ KREX proteins cloned in frame with BCCP, which acts as a folding marker and solubility enhancer

▪ Biotin added to cells and attached to correctly folded proteins by cellular biotin ligases

▪ Misfolded proteins washed away as they drive co-translational misfolding of BCCP, resulting in loss of function of BCCP

▪ Only correctly folded proteins are attached through binding between BCCP


KREX protein expression is highly efficient6

▪ KREX technology aids protein folding (more proteins are solublyexpressed in BCCP pMD004 vector compared to pQE-GFP-biotin vector)

▪ >98% of proteins were expressed solubly when using KREX

▪ Whereas conventional approaches only give 48% success rate

▪ Mechanism based on increasing solubility of proteins that may have previously been targeted for degradation or form inclusion bodies

Protein Expression Summary. Proteins were chosen and expressed into pQE-GFP-biotin (expression

vector 1) and BCCP pMD004 (expression vector 2). Expression of the proteins are highlighted and

represented as:C – cloned but not expressH– expressing hexa-histidine positive protein in SDS-PAGE Western blotB – expressing biotin positive protein in SDS-PAGE Western blot.S – expressing soluble protein.

0

20

40

60

80

100

98.8%

expression

success rate


Unique surface chemistry of KREX protein arrays

▪ BCCP-tagged, biotinylated KREX proteins are immobilised onto customisedstreptavidin-coated hydrogel

▪ KREX proteins retain folded structure and function in an aqueous environment

▪ PROTEINS behave as if they are in free solution

▪ Simultaneous purification and immobilisation - “Surface Capture”

7

COO-

COO-

COO-

COO-

COO-

COO-

Glass slide

Hydrogel layer

Streptavidin

tetramers covalently bound to hydrogel layer

BCCP-tagged

proteins

▪ Single attachment point - consistent orientation

▪ 50 Angstrom spacer - protein projected in aqueous environment

▪ Very high binding affinity & specificity▪ Biotin : avidin (KD = 10-15 M)

▪ Antigen : antibody (KD = 10-9 M)

▪ His tag : Ni-NTA (KD = 10-6 M)


Proteins on KREX arrays have been functionally validated

▪ Kinases auto-phosphorylate and Kinase inhibition assays correlates with published data

▪ DNA binding proteins bind DNA – p53 mutant proteins exhibit differential binding of labelled oligo

▪ Indirect functional assays

8

Kinases autophosphorylatein the presence of ATP

P53 mutants bind labelled DNA oligo with different Kd values

Autoimmune disease hits correlate with published data


KREX arrays exhibit exceptional sensitivity and specificity9

Other array

Sengenics

1/128,000

folddilution

1 part in 100 billion

Other arrays have high background due to non-specific interactions of antibodies to with misfolded

hydrophobic proteins

Highly reproducible duplicates performance giving Pearson Correlation >0.99

Five orders of magnitude dynamic range

Exceptional sensitivity10pg/ml range

Single digit µl levels of sample can be used


Our products10

• Contains over 1600 KREX™ proteins spotted in quadruplicate• Broad coverage: includes kinases, signalling molecules, cytokines,

interleukins, chemokines and cancer antigens• Picogram level sensitivity• Fully quantitative arrays with exceptional consistency

Immunome Discovery

Array

• Contains over 300 KREX™ Kinase proteins spotted in triplicate• Picogram level sensitivity• Fully quantitative arrays with exceptional consistency

• The array can be customised with any protein, any number of proteins, any species and format (16-plex, 8-plex, 4-plex, 2-plex and single format).

• Sengenics only offers immobilised proteins onto a specified solid support format for the KREX™ Custom Array.

Custom Arrays

Human Kinome

Array


Kinase solutions with KREX11

▪ The cost of taking a drug to the market today is ~ $1.5bn

▪ 9 out of 10 compounds fail clinical trials

▪ ~ 50% of failures due to side effects

▪ Suggests that new technological means to quantify drug binding and selectivity could have many benefits:

▪ Improving selectivity of drug candidates in development

▪ Identifying targets of hits/leads arising from phenotypic screens

▪ Identifying off-targets that are responsible for adverse drug reactions

▪ Improving understanding of poly-pharmacology

▪ Identifying new targets from old drugs

▪ Between 2000 and 2015, the US FDA approved a total of 361 NMEs with only a total of 479 targets

▪ Number of targets per approved drug suggests that selectivity of existing drugs is also relatively poor - In all of the 361 approved NMEs, 40% are single-target drugs

▪ Only small number of NMEs have a target number ≥ 5

[Adapted from Nature Scientific Reports vol. 7, Article number: 12230 (2017)]

Discovery

Analysis

Validation

Assay

Lead

Phase I

Phase II

Phase III

FDA Approved

10,000 Compounds

1,000 Compounds

20 Compounds

11 Compounds

5 Compounds

3 Compounds

1 Drug


The role of kinases in drug discovery12

▪ Protein kinases are involved in every aspect of normal and pathological cell physiology

▪ Over 400 human diseases are linked to defects in kinases-dependent signaling pathways:

▪ Under pathological conditions Kinases can be deregulated or mutated

▪ Any variations in their phosphorylation states could result in biological abnormalities

▪ Ideal targets in drug discovery!

▪ There are 518 kinases encoded in the human genome – Sengenics currently has 336 kinases expressed using KREX

Nature Reviews Drug Discovery volume 17, pages 353–377 (2018)

Kinase inhibitors in diverse biological processes and new therapeutic areas

FDA-approved kinase inhibitors for oncology and non-oncology indications (autoimmune and inflammatory disease)


The role of kinases in drug discovery13

▪ KREX Kinome Array

▪ Kinase profiling studies have shown that potential inhibitors tend to bind to kinases that are distributed throughout the kinome phylogenetic tree

▪ Therefore, the ability to profile the entire kinome simultaneously is advantageous in drug discovery

▪ Eukaryotic kinases have strong structural homology in their catalytic domains: Likely leads to downstream difficulties with selectivity for drugs that inhibit kinases

▪ Sengenics currently has 336 kinases expressed using KREX, immobilised onto customised streptavidin-coated hydrogel surface which retain folded structure and function in an aqueous environment

▪ The platform allows for the quantitative and reproducible measurement of the binding of inhibitors to the human kinome, using only nanolitre quantities of kinase crude lysates

COO- COO- COO- COO- COO- COO-

Glass slide

Hydrogel layer

Streptavidin tetramers

covalently bound to hydrogel layer

BCCP-tagged proteins


Applications – Kinase auto-phosphorylation

▪ Inhibition of Kinase Auto-phosphorylation

▪ Platform: Sengenics 100+ Kinome Array

▪ Results:

▪ This study demonstrates that human kinases on the array are present as homodimers (required for catalytic activity, as evidence by autophosphorylation in the presence of ATP)

▪ Kinase inhibition of auto-phosphorylation by small molecule inhibitors can be quantified

▪ Kinase activity correlates with published solution phase data

14

ATP + Fes kinasecontrol (ATP only)

10 µM ATP

Kinase buffer only

10 µM ATP + 10 µM staurosporine

1 - Incubate with inhibitor

ATP

ATP

2 - Add ATP, incubate and wash

P

P

3 - Quantitative autophosphorylation

P

P

4 - Detect with labelled anti-P ab


Applications – Kinase auto-phosphorylation

▪ Inhibition of Kinase Auto-phosphorylation

▪ Platform: Sengenics 100+ Kinome Array

▪ Results:

▪ This study demonstrates that human kinases on the array are present as homodimers (required for catalytic activity, as evidence by autophosphorylation in the presence of ATP)

▪ Kinase inhibition of auto-phosphorylation by small molecule inhibitors can be quantified

▪ Kinase activity correlates with published solution phase data

15

1 - Incubate with inhibitor

ATP

ATP

2 - Add ATP, incubate and wash

P

P

3 - Quantitative autophosphorylation

P

P

4 - Detect with labelled anti-P ab


Applications: On-array kinase inhibitor binding assays

▪ How do you build a generic array-based assay for > 300 kinases when they don’t all auto-phosphorylate, have different enzymatic mechanisms and different substrates, inhibitors aren’t fluorescent and can’t be easily be modified without affecting SAR?

▪ Solution: Build competitive displacement assays using a universal kinase probe:

▪ Biotinylated photo-labile ATP analog is a broad-specificity kinase probe

▪ Incubate kinase protein microarray with varying concentration of photoaffinity label

▪ Photolyse with UV irradiation (254nm )

▪ Detect labeled kinases with fluorescently-labeled streptavidin

▪ Quantify on microarray scanner

16

Biotinylated 8-azido-ATP analog

CDK2 with bound peptide

substrate

Phosphorylase Kinase with

bound peptide substrate

Biotinylated 8-azido-ATP analog



▪ Binding of 8-azido-ATP analog to each arrayed kinase is concentration dependent and saturable

17

KINASE Kd (μM) R squared (%)

AKT1 43±2 94±3

CDK2 35±5 95±2

CDK4 37.5±3.5 96±3

CDK7 45±2 93.5±3.5

FES 32±4 94.5±3.5

FGFR2 21.5±3.5 95.5±2.5

MAPK1 68±2 94±2

MAPK3 55.5±14.5 96±0

PRKCZ 46±4 92.5±0.5

RAF 42.5±4.5 92.5±1.5

SRC 30.5±1.5 91.5±1.5

EGFR 36.5±1.5 96±0

pNP400[EGFR Wt]

0 25 50 75 100 125 1500

2500

5000

7500

10000

ABP concentration (uM)

RF

U

AKT1[B]

0 25 50 75 100 125 1500

10000

20000

30000


RF

U

CDK2[B]

0 25 50 75 100 125 1500

5000

10000

15000


RF

U

SRC[B]

0 25 50 75 100 125 1500

2500

5000

7500

10000


RF

U

CDK4[B]

0 25 50 75 100 125 1500

5000

10000

15000


RF

U

CDK7[B]

0 25 50 75 100 125 1500

10000

20000

30000


RF

U

FES1[B]

0 25 50 75 100 125 1500

10000

20000


RF

U

FGFR2 1[B]

0 25 50 75 100 125 1500

2500

5000

7500


RF

U

MAPK1[B]

0 25 50 75 100 125 1500

10000

20000


RF

U

MAPK3[B]

0 25 50 75 100 125 1500

25000

50000

75000


RF

U

PRKCZ[B]

0 25 50 75 100 125 1500

2500

5000

7500


RF

U

RAF[B]

0 25 50 75 100 125 1500

2500

5000

7500


RF

U

pNP400[EGFR Wt]

0 25 50 75 100 125 1500

2500

5000

7500

10000


RF

U

AKT1[B]

0 25 50 75 100 125 1500

10000

20000

30000


RF

U

CDK2[B]

0 25 50 75 100 125 1500

5000

10000

15000


RF

U

SRC[B]

0 25 50 75 100 125 1500

2500

5000

7500

10000


RF

U

CDK4[B]

0 25 50 75 100 125 1500

5000

10000

15000


RF

U

CDK7[B]

0 25 50 75 100 125 1500

10000

20000

30000


RF

U

FES1[B]

0 25 50 75 100 125 1500

10000

20000


RF

U

FGFR2 1[B]

0 25 50 75 100 125 1500

2500

5000

7500


RF

U

MAPK1[B]

0 25 50 75 100 125 1500

10000

20000


RF

U

MAPK3[B]

0 25 50 75 100 125 1500

25000

50000

75000


RF

U

PRKCZ[B]

0 25 50 75 100 125 1500

2500

5000

7500


RF

U

RAF[B]

0 25 50 75 100 125 1500

2500

5000

7500


RF

U



▪ Photo-labile kinase probe enables quantitative inhibitor binding assays to be built for unlabelled compounds

▪ On-array competition binding assays give inhibitor binding constants for unlabelled kinase inhibitors directly

18

KINASER squared

%

Binding

Constant (Kd)

for ATP (μM)

Inhibition

constant Ki for

staurosporine

(nM)

Inhibition

constant Ki for

PKC-412 (nM)

AKT1 97 242 41 476

CDK2 99 260 23 1124

CDK4 99 179 41 1222

CDK7 98 299 24 1211

FES 94 288 36 964

FGFR2 92 131 30 646

MAPK1 97 445 74 1522

MAPK3 96 256 53 1343

PRKCZ 89 196 23 564

RAF 92 291 61 1039

SRC 98 187 15 735

EGFR wt 94 267 37 829

Ki = IC50 / {1 + [FL]/Kd}

COMPETITION BINDING ASSAYOF EGFR pNP200 WITH

STAUROSPORINE

0 1 2 3-50

0

50

100

150

Log[staurosporine] nM

No

rmali

sed

RF

U

COMPETITION BINDING ASSAYOF CDK4 WITH

STAUROSPORINE

0 1 2 3-50

0

50

100

150

Log[staurosporine]nM

No

rmali

sed

RF

U


Applications: On-array Drug selectivity

▪ Arrays of 100+ human kinases probed with Staurosporine and Iressa using competitive displacement assay

▪ Percentage inhibition of 8-azido-ATP analog (40μM) binding to kinases on the microarray in the presence of a single Staurosporine or Iressa concentration (1μM)

▪ 47 candidate targets for Iressa identified (inc EGFR)

19

Blackburn et. al. Methods in Molecular Biology, vol. 785. 2011. 305–30

Kinase % Inhibition p-value Kinase % Inhibition p-value

ADRBK2 85.3±0 0.01 MGC42105 90.0±1 0.01 AURKB 89.6±0 0.00 MKNK1 91.7±1 0.00 BMX 75.3±3 0.04 PDK4 28.6±7 0.00

C20orf64 / PRPK 60.2±4 0.00 PMVK 28.7±14 0.01 CAMKK2 28.9±10 0.00 PRKACB 32.0±6 0.01 CDC2 77.9±1 0.04 PRKCB1 17.0±4 0.00 CDC2L1 44.1±23 0.02 PRKCH 90.7±0 0.03 CDK4 80.4±2 0.00 PRKCN 90.4±0 0.00

CDK5 67.3±5 0.00 PRKD2 90.5±0 0.00 CHEK2 87.4±1 0.00 PTK7_aa 31-704 70.8±1 0.00 CLK1 46.7±6 0.00 PTK7_aa 726-1070 86.0±0 0.00 CLK2 28.5±12 0.02 RAGE 79.6±7 0.01 COL4A3BP 89.5±1 0.04 RBKS 42.6±1 0.05

CSNK1G1 59.0±4 0.00 RET 83.0±2 0.00 DYRK4 63.6±5 0.00 RIOK3 70.0±2 0.00 EGFR_aa 669-1210 46.7±4 0.02 RPS6KA1 95.4±0 0.00 H11 74.6±1 0.04 STK18 84.8±0 0.00 ITPKB 86.7±2 0.00 STK25 85.9±1 0.01

MAPK11 71.7±2 0.00 STK3 78.4±1 0.03 MAPK3 47.8±4 0.01 STK33 65.3±2 0.03 MAPK7 46.9±2 0.02 STK38 76.1±1 0.01 MAPK8_2 80.6±1 0.04 TK1 84.3±2 0.03 MATK 64.6±4 0.00 TYRO3_aa 451-890 83.2±1 0.05

MGC16169 38.8±3 0.01

Staurosporine

Iressa


Applications: predictive toxicology and kinase mutants

▪ On-array competition binding assays allow inhibitor binding constants for Iressa to be measured directly on array

20

Clinically relevant mutations in EGFR catalytic domain

affect drug binding

COMPETITION BINDING ASSAYOF FES WITH IRESSA

0 1 2 3 4 5-50

0

50

100

150

Log[IRESSA]nM

No

rm

ali

sed

RF

U[%

]

COMPETITION BINDING ASSAYOF SRC WITH IRESSA

0 1 2 3 4 5-50

0

50

100

150

Log[IRESSA]nM

No

rm

ali

sed

RF

U[%

]

COMPETITION BINDING ASSAYOF EGFR pNP200 WITH IRESSA

0 1 2 3 4 5-50

0

50

100

150

Log[IRESSA]nM

No

rm

ali

sed

RF

U[%

]

Iressa

Ki (nM) (literature)

Iressa Ki (nM) (array)

Erbstatin Ki (nM) (array)

FES >10,000 2.70 -

SRC 3800 2.40 36

EGFR wt 1 2.47 61

EGFR (L858R) 0.94 2.20 59

EGFR (G719S) 1.1 2.36 69

EGFR DE746-A750) 0.54 1.55 78


Applications: label-free detection of protein-small molecule interactions

▪ MALDI mass spectrometry readable protein microarrays enable protein ID on-chip

▪ > 10,000 samples/set

▪ attomol sensitivity

▪ protein purification on chip

▪ On-probe capture and identification

▪ 3nl sample volume

▪ desorption of intact proteins

▪ Protein tryptic digest fingerprint of arrayed proteins

▪ small molecule binding

▪ enzymatic catalysis

21

[M+H]+ GST avidin

770.416 X919.52 X

963.497 X1026.563 X

1032.574 X

1094.56 X

1138.518 X1149.628 X1182.645 X1314.736 X1516.828 X2326.1 X845.5 X2211.1

2283.094 X919.53 X

1235.48 X

1595.845 X

2003.02 X

trypsin

X



▪ MALDI mass spectrometry readable protein microarrays enable protein-small molecule interactions to be measured on-chip

▪ Array-based ligand binding monitored directly by MALDI mass spectrometry

▪ Affinity of peanut agglutinin for lactose is in the micromolar range and is easilydetectable by on-chip mass spectrometry

22

Protein-ligand interactions identified directly by mass spectrometry

Rh-lactose

Bound to PNA

Bound to FKBP12



▪ MALDI mass spectrometry readable protein microarrays

▪ Generic kinase assay on a biochip: quantification of interconversion of ATP and ADP quantified by laser desorption mass spectrometry

▪ Each reaction is encapsulated in a nanodroplet sitting over the arrayed kinase

▪ Enables on-chip assessment of catalytic turnover by kinases and therefore in principle allows on-chip measurement of inhibition of turnover by drug candidates

23

ADP ATP

+Na

+2Na

+Na

ATP formation from ADP and phosphocreatine, catalysed by creatine kinase


Applications: custom proteins and arrays

▪ Any number of proteins

▪ Any proteins

▪ Any species

▪ Any expression systems

24


Applications: on-array protein-x interaction

▪ Eliminating the restrictions of direct labelling of drug molecules:

▪ Individual drug profiling against the human kinome:

▪ Incubate kinome array with fluorescently-labelled drug compounds / molecules

▪ Vary concentrations of drug compounds / molecules to determine the binding affinities

▪ Scan and Quantify

25

Cy3Cy3 Cy3


MDM2p/His ratio

0

0.2

0.4

0.6

0.8

1

1.2

wt

W23A

W23G

R72P

P82L

M133T

Q136X

C141Y

P151S

P152L

G154V

R175H

E180K

R181C

R181H

H193R

R196X

R209X

R213X

P219S

Y220C

S227T

H233N

H233D

N235D

N235S

S241F

G245C

G245S

G245D

R248W

R248Q

I251M

L252P

T256I

L257Q

E258K

L265P

V272L

R273C

R273H

P278L

R280K

E286A

R306X

R306P

G325V

R337C

L344P

S392A

Applications: On-array Protein-X interaction

▪ Protein-Protein Interaction Study

▪ Quantifying the effect of clinically-relevant mutation on the MDM2 binding properties of p53

▪ p53 array probed with Cy3-labelled MDM2 [top]

▪ p53 lysate incubated with FLAG-MDM2 lysate and subjected to a FLAG-agarose pull down assay [bottom]

▪ Results:

▪ WT phenotypes were observed for all mutants except the negative control.

▪ The assay demonstrated the feasibility of the format for investigating other p53-protein interactions and offers some advantages over the commonly used two-hybrid methods, including increased throughput and control over binding conditions (e.g., concentrations and buffers)

26

MDM2 + - + -WT W23A

WT

W2

3A

InputLysates


Applications: On-array Protein-X interaction

▪ DNA-Protein Interaction Study

▪ Study Design : p53 microarray probed with Cy3-labelled GADD45 duplex oligo

▪ Results:

▪ Quantitative parallel thermodynamic analysis revealed previously unknown functional effects of mutation on p53 protein-DNA interaction

▪ Mutations outside of the DNA-binding domain generally had near wild-type affinity for DNA.

▪ The quantitative analysis allowed the functional classification of mutants into 4 groups:

▪ those with wild-type affinity;

▪ those exhibiting reduced stability;

▪ those with reduced affinity; and

▪ those showing complete loss of activity

27

Boutell et. al. Proteomics 2004, 4, 1950–1958


Applications: KREX proteins can then be attached to a wide variety of surfaces28

M I C R O A R R A Y S B I A C O R E M A S S S P E CM I C R O T I T R E P L A T E S


Summary

▪ KREX Technology:

▪ unique platform

▪ proteins are correctly folded and functional with 98% success rate

▪ exceptional consistency and high accuracy

▪ can be customised for additional proteins in various expression systems

▪ The Role of Kinases in Drug Discovery:

▪ Over 400 human diseases are linked to defects in kinases-dependent signaling pathways

▪ Ideal targets in drug discovery

▪ Sengenics currently has 336 our of the 518 correctly folded, functional kinases in its library

▪ The KREX Kinome Array

▪ Allows profiling of the entire kinome simultaneously

▪ KREX produced kinases retain their folded structure and function in an aqueous environment

▪ Highly sensitive and precise: the platform allows for the quantitative and reproducible

measurement of kinase interactions in the nanoliter range

29

Sengenics UK Ltd. Registered in England and Wales no: 11016361

Sengenics Corporation Pte Ltd. Registered in Singapore : no 201734100D

Email: [email protected]

Tel: +44 (0)1223 926 588

Documents

Applications of KREX for Kinases€¦ · A BP c o ne tr a i ( u M) R F U CDK2 [B] 0 25 75 100 125 150 5000 10000 15000 A BP c o ne tr a i ( u M) R SR C [B ] 0 25 50 75 100 125 150