TAO LINSenior Application Specialist

What is LabChip Technology?

Miniaturization

Integration

Automation

Active Fluidic Control Pressure and vacuum Electrokinetics

Automated Data analysis Quantitation, Sizing, Quality Metrics…

…allows you to assemble the power of a laboratory full of people and equipment on a single chip.

Microfluidic Chips

Kinase / ATP

Reactions in 96/384 well plate – Read on EZReader

4- and 12-sipper Microfluidic Mobility Shift Chips

Upstream Electrode Well

Fused Silica Sipper

DownstreamElectrode

Well

Detection Window

What Does LabChip Data Look Like?

…like peaks on a chromatogram

The product andsubstrate are separatedand shown as two peaks

Product

Substrate

What’s EZ Reader?系统功能：针对各种酶类及 GPCR类药靶的化学或天然药物的筛选及机理研究

1 ）酶类药靶：底物及产物有荷电变化；底物为（或可设计为）多肽或其他小分子2 ） GPCR ：需配合 PathHunter eXpress Assay 使用，现有 28 种， more in development

原理：• 基于微流体芯片技术原理，通过 Mobility Shift Assay对 反应底物及产物进行分离及直接检测；• 具备终点法及实时动力学两种检测模式

Features of Mobility Shift LabChip Assays

1 ）先分离后检测：通过芯片将一个反应的底物及产物分开并分别检测其荧光强度；2 ）直接检测：整个体系中不涉及任何抗体或其他中间体，而是直接检测反应底物 及产物；3 ）微量化： 96/384 孔板反应，亚 nM 级酶浓度，微量反应体系（ 4-12 ），微量上样4 ）具备实时动力学检测模式：高效、经济，且避免了孔与孔之间的误差；5 ） ATP不依赖：可以容忍很宽的 ATP 浓度范围；

实验结果准确、可靠、灵敏，

即便很弱的药物作用也可被准确检出，

系统 Z’>0.9

• Multiple Target Classes： Protein Kinases Kinase Cascades Lipid Kinases Protein Phosphatases Protease Lipid Modifying Enzymes Histone Deacetylases

(HDAC) & Sirtuins Histone Demethylase Phosphodiesterases (PDE) Acyl-Transferases…… GPCRs

Multiple Application Fields ： In-house Assay Development Primary Screening （ +pooling ） Secondary Screening/

Hit Confirmation Mechanism of Action Kinase Profiling/

(with ProfilerPro Kits)

Features of Mobility Shift LabChip Assays

Linearity of Reactions

Kinase MOA Studies(PKA)Kinase MOA Studies(PKA)• Part of Data by Jeff Hirsch, Matt Saabye, Holly Hall & Hoe Monahan, Pfizer Global R&D, St.Louis

Kinase MOA Studies(PKA)Kinase MOA Studies(PKA)• Testing for Reversible Binding• Determine whether enzyme activity recovers upon rapid dilution of

enzyme-inhibition complex

100×Enzyme ＋ Compound at 10×IC50

Incubate

Dilute 100-fold into reaction buffer

containing 1×substrate

Generate progress curve

(91% Inhibition)

(9% Inhibition)

Kinase MOA Studies(PKA)Kinase MOA Studies(PKA)• Testing for Reversible Binding• Both H89 and Staurosporine are reversible binding to PKA.

Kinase MOA Studies(PKA)Kinase MOA Studies(PKA)Testing for Reaction LinearityGenerate progress curves from reactions initiated by addition of enzyme to wells containing substrates and inhibitor

Kinase MOA Studies(PKA)Kinase MOA Studies(PKA)• Steady-State Analysis of Inhibition Mechanism

ATP-Competitive Inhibitionof PKA by H89

-4 -3 -2 -1 0 1 2 3

0

20

40

60

80

100

Log [H89] (M)

% In

hibi

tion

Non-Competitive Inhibitionof PKA by PKI 6-22

-6 -5 -4 -3 -2 -1 0

0

25

50

75

100

2000 500 125 31.3 7.81 1.95

[ATP] (M)Log [PKI] (M)

% In

hibi

tion

1 10 100 1000

0

1

2

3

4

5

6

7

[ATP] (M)

IC50

(

M)

1 10 100 10000

1

2

3

4

5

6

7

[ATP] (M)

IC50

(nM

)

Kinase MOA Studies(PKA)Kinase MOA Studies(PKA)

Peptide-Competitive Inhibitionof PKA by PKI 6-22

-6 -5 -4 -3 -2 -1 0

0

20

40

60

80

100

Log [PKI] (M)

% I

nh

ibit

ion

Non-competitive Inhibitionof PKA by H89

-4 -3 -2 -1 0 1 2

0

20

40

60

80

100

7.4 2.4 0.82 0.27 0.09

[Peptide] (M)Log [H89] (M)

% I

nh

ibit

ion

0.1 1 100

1

2

3

4

5

6

[Peptide] (M)

IC5

0 (n

M)

0.1 1 10-1

0

1

2

3

4

5

[Peptide] (M)

IC5

0 (

M)

Steady-State Analysis of Inhibition Mechanism

Kinase MOA Studies(PKA)Kinase MOA Studies(PKA)

• Steady-State Analysis of Inhibition Mechanism• H89 is ATP-competitive while PKI6-22 is peptide-competitive

Kinase MOA StudiesKinase MOA Studies• Compound Off-Rates

T1/2 value determination of compounds against a Ser/Thr kinase

Figure 8. Inhibitor and enzyme were pre-incubated (50 nM enzyme and 50 nM inhibitor) for 1 hour at room temperature. The pre-incubation mixture was diluted into saturating amounts of MgATP (40X Km) to provide a final concentration of 0.1 nM enzyme and 0.1 nMinhibitor, respectively. The dissociation rates (koff) were determined using Equation 4.

Time(min)

0 20 40 60 80 100 120 140 160 180 200

Pro

du

ct (n

M)

0

100

200

300

400

500

Preincubation W/O inhibitor

Compound 1 (t1/2 = 34 min)

Compound 2 (t1/2 = 1.4 hrs)

Compound 3 (estimated t1/2 = 5hrs)

Compound 4 (estimated t1/2 = 8 hrs)

Compound 5 ( t1/2 >> 3 hrs)

T1/2 value determination of compounds against a Ser/Thr kinase

Figure 8. Inhibitor and enzyme were pre-incubated (50 nM enzyme and 50 nM inhibitor) for 1 hour at room temperature. The pre-incubation mixture was diluted into saturating amounts of MgATP (40X Km) to provide a final concentration of 0.1 nM enzyme and 0.1 nMinhibitor, respectively. The dissociation rates (koff) were determined using Equation 4.

Time(min)

0 20 40 60 80 100 120 140 160 180 200

Pro

du

ct (n

M)

0

100

200

300

400

500

Preincubation W/O inhibitor

Compound 1 (t1/2 = 34 min)

Compound 2 (t1/2 = 1.4 hrs)

Compound 3 (estimated t1/2 = 5hrs)

Compound 4 (estimated t1/2 = 8 hrs)

Compound 5 ( t1/2 >> 3 hrs)

Kinase Profiling

Why Kinome profiling?Why Kinome profiling?518 Protein kinase genes

>150 kinases implicated in disease

>120 kinases associated with cancer

Drug approved Target disease

Gleevec (2001) TK CML, GISTIressa (2003) TK NSCLCTarceva (2004) TK NSCLC

pancreatic cancer

Nexavar (2005) S/TK RCCSprycel (2006) TK CML Sutent (2006) TK GIST, RCCTykerb (2007) TK Breast, Lung cancer

>70 inhibitors currently in clinical trials for cancer

Most screened target in drug discovery in 20072 (23% HTS)

1. Kinome Dendrograms from Manning G et al, Science 1912-1934(2002) and Cell Signaling Technology 2. Downey and Shauna, Drug Discovery World Fall 2007, 79-84,

EZ Reader and ProfilerPro Kits

• Complete solution for kinase profiling – ProfilerPro kits, microfluidic chip & EZR– Validated assays– High data quality

• Rapid in-house profiling– Compounds remain on site– Same day results

• Low cost solution– Competitive cost per well vs. outsourcing

ProfilerPro Kits for Kinase ProfilingProfilerPro Kits for Kinase ProfilingKinase Profiling:1 compound vs the whole/partial kinome

Find possible target of compounds Target selectivity of hits Better & efficient structure modification

ProfilerPro Kits: All-in-One, Ready-to-Use

• Enzyme plate– 24 kinases pre-dispensed per

plate

• Peptide substrate/ATP plate– ATP at apparent Km – Full and no activity controls

included

• All required buffers – Reconstitution Buffer– Termination Buffer

• Initially 2 kits available– 48 different kinases

• Additional kinases being added

• Custom enzyme plates possible

ProfilerPro Kits

Up to 12 compounds can be assayed in one plate !

Plate Layout• Three no-inhibitor control rows (A,B and O)• One 100% inhibition control row (P)

– No ATP• 12 compound rows

ProfilerPro assays have broad kinome coverage

PP 1 Kinase Family1 MAPKAPK2CAMK2 AurA Atypical3 PKCz AGC4 Rsk1 CAMK5 PRAK(MAPKAPK5)CAMK6 Erk1 CMGC7 PKD2 CAMK8 CK1d CK19 CHK1 CAMK

10 Abl TK11 Fyn TK12 LynA TK13 CHK2 CAMK14 Met TK15 Lck TK16 Src TK17 Gsk3b CMGC18 Erk2 CMGC19 PKA AGC20 Akt2 AGC21 INSR TK22 p38a CMGC23 Akt1 AGC24 Msk1 CAMK

PP2 Kinase Family25 PKCb2 AGC26 ROCK2 AGC27 CDK2 CMGC28 MST2 STE29 PKG1a AGC30 PAK2 STE31 IGF1R TK32 FGFR1 TK33 MARK1 CAMK34 CamK2d CAMK35 Pim2 CAMK36 BTK TK37 cTAK1 TKL38 Dyrk1a CMGC39 CamK4 CAMK40 AMPK CAMK41 Flt3 TK42 HGK TK43 VEGFR2(KDR)TK44 cRAF(Raf1)TKL45 p70S6K AGC46 IRAK4 TKL47 SGK1 AGC48 SyK TK

Now 210+

PP3 Kinase Family49 AurB Atypical50 FGFR2 TK51 FGFR3 TK52 Abl(Q252H)TK53 AurC Atypical54 FGFR4 TK55 EGFR TK56 Abl(T315I) TK57 IKKb Atypical58 MAPKAPK3CAMK59 p38b2(MAPK11)CMGC60 TSSK1 CAMK61 PKG1b AGC62 CaMK2b CAMK63 p38d CMGC64 TSSK2 CAMK65 Abl(H396P)TK66 PDGFRa TK67 FGFR2(N549H)TK68 Hck TK69 Flt3(D835Y)TK70 Fer TK71 Akt3 AGC72 CamK2g CAMK

PP4 Kinase Family73 AKT3 AGC74 MSK2 (RPS6KA4)CMGC75 NEK2 CAMK76 Mark2 CAMK77 BMX TK78 CSNK1A1 (CK1a)CK179 BRSK1 CAMK80 BRSK2 CAMK81 PKD1 Atypical82 PhKg1 CAMK83 SGK2 AGC84 SGK3 (SGKL)AGC85 ARG TK86 DCAMKL2 CAMK87 RSK2 AGC88 RSK3 AGC89 PKD3 CAMK90 PKC-alpha AGC91 PKC-beta1 AGC92 PKC-gammaAGC93 PIM1 AGC94 PKC-delta AGC95 PKC-epsilonAGC96 PKC-theta AGC

PP 5 Kinase Family97 EPHA1 TK98 EPHA2 TK99 EPHA3 TK

100 EPHA4 TK101 EPHA5 TK102 EPHB2 TK103 EPHB3 TK104 EPHB4 TK105 DYRK1B CMGC106 LYNB TK107 GCK (MAP4K2)STE108 MINK (MINK1)CAMK109 ABL1 (E255K)TK110 FGR TK111 MST1 (STK4)TK112 FLT1 TK113 ABL1 (Y253F)TK114 FES TK115 FLT4 TK116 TEC TK117 ABL1 (G250E)TK118 LTK TK119 FMS (CSF1R)TK120 HER4 (ERBB4)TK

PP6 Kinase Family121 ROCK1 AGC122 PASK CAMK123 PhKg1 CAMK124 Yes TK125 PIM3 CAMK126 PhKg2 CAMK127 DCAMKL1 CAMK128 EGFR (T790M)TK129 DYRK3 CMGC130 DYRK4 CMGC131 CLK2 CMGC132 MST1R TK133 HIPK1 CMGC134 HIPK2 CMGC135 RSK4 AGC136 PDGFR-a(V561D)TK137 EPHA8 TK138 CDK5/p25 CMGC139 BLK TK140 ALK TK141 PYK2 (PTK2B)TK142 DAPK1 CAMK143 CK 1g2 CK1144 FRK TK

PP7 Kinase Family145 JAK2 TK146 ROS (ROS1)TK147 RET TK148 EPHB1 TK149 FGFR3 (K650E)TK150 EGFR (T790M L858R) TK151 RET (Y791F)TK152 TXK TK153 ITK TK154 TYRO3 TK155 CaMK2a CAMK156 KIT TK157 TRKC (NTRK3)TK158 Mer TK159 CK1g3 (CSNK1G3)CK1160 MET (M1250T)TK161 AXL TK162 MARK4 CAMK163 MELK CAMK164 CDC2/Cyclin B1CMGC165 KIT[T670I] TK166 AMPK-a2/b1/g1CAMK167 PRKCI (PKC-iota )AGC168 ZIPK (DAPK3)CAMK

PP8 Kinase Family169 Ret (V804L)TK170 SRM (SRMS)TK171 PRKX AGC172 GSK3-alphaCMGC173 FGFR1-(V561M)TK174 NTRK2 (TRKB)TK175 p38alpha (T106M)CMGC176 DDR2 TK177 CK1-epsilonCK1178 MST3 (STK24)STE179 PDGFRA (D842V)TK180 NuaK1 CAMK181 CK1-g1 (CSNK1G1)CK1182 NEK1 Atypical183 PDGFRβ TK184 MNK1 (MKNK1)CAMK185 PAK4 STE186 CaMK1a CAMK187 PAK3 STE188 IKBKE (IKK epsilon)Atypical189 PAK5 (PAK7)STE190 Camk1d CAMK191 LOK STE192 CDK3 CMGC

193 ACK (TNK2)194 CK21 (CSNK2A1)195 CK22 (CSNK2A2)196 CLK3197 DYRK2198 EGFR (L858R)199 EPHA6200 EPHA7201 HIPK3202 IRAK1203 JAK3204 MAP4K5 (KHS1)205 MARK3206 Met (Y1235D)207 MNK2(MKNK2)208 MST4209 P70S6K (RPS6KB2)210 PAK6211 SRC (T341M)212 SRPK2213 TAOK3214 TEK (Tie2)215 TRKA (NTRK1)

Profiler Pro Kinome Coverage

ProfilerPro assays are validated during development

(B) Reaction linearity as a function of [E]

0

0.05

0.1

0.15

0.2

0.25

0.3

0 0.5 1 1.5 2

time (hours)

fra

ctio

na

l co

nve

rsio

n

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 5 10 15 20 25 30

[CHK2] nM

fra

ctio

na

l co

nve

rsio

n a

t 1 h

r.

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0 1 2 3 4 5 6

% DMSO

rate

( u

M/h

ou

r)

CHK2-AT P Km

0 250 500 750 1000 12500.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009R1R2R3

[ATP] (uM)

Re

ac

tio

n R

ate

(u

M/m

in)

(A) ATP Kmapp of 57.8 ± 10 uM

(C) Reaction linearity with time (D) Effect of DMSO on the reaction rate

ATP Kmapp = 57.8 ± 10 uM

Sample Assay Development Data for CHK1

13 standard kinase inhibitors were tested at [10 uM]

AK

T1

MS

K1

GS

K3b

Erk1

AK

T2

CK

1d

INS

R

ER

k2

p38

ME

T

CH

K1

Src

AB

L

RS

K1

CH

K2

FY

N

LCK

PK

Cz

PR

AK

AurA

LYN

MK

2

PK

D2

PK

A

Bis-I 89 95 94 19 66 33 9 19 19 59 80 36 8 98 78 51 64 77 49 51 38 46 92 82

H-89 90 96 -4 25 69 29 3 20 -3 20 62 18 26 65 57 6 40 14 60 58 16 58 85 99

staurosporine 98 100 97 84 99 54 90 84 23 100 99 100 94 99 99 100 99 91 96 100 99 97 98 100

go6976 73 97 94 82 90 71 4 90 64 93 99 79 79 96 86 98 83 30 94 97 88 88 98 98

PP2 3 24 22 24 1 90 5 25 56 -4 17 99 89 17 22 100 95 -10 61 53 98 28 74 96

SB203580 2 8 86 29 -10 90 -2 18 97 38 -3 61 24 -1 -4 40 48 -22 57 19 47 1 55 62

K252a 85 99 95 99 63 38 68 100 -4 99 100 98 59 99 100 97 95 34 98 100 98 97 99 100

SU6656 4 17 54 0 28 51 7 13 -6 45 43 94 65 43 51 92 80 -16 56 96 86 61 47 82

5iodotubericidin 2 29 53 92 6 94 33 92 21 40 14 49 43 47 45 41 45 30 66 64 42 42 97 77

H-7 4 29 24 7 1 19 3 4 19 31 -10 9 32 37 14 21 0 -4 66 17 -10 23 82 78

H-9 56 91 93 42 28 42 10 46 36 59 64 8 65 77 47 20 15 61 79 43 17 71 97 90

KN-62 50 80 91 -34 91 67 -3 35 -3 91 37 86 74 33 62 100 47 10 96 90 74 97 95 87

quercetin 94 84 94 92 98 89 12 99 26 96 -7 99 69 94 72 100 51 -418 95 100 98 96 95 100

AK

T1

MS

K1

GS

K3

Erk1

AK

T2

CK

1d

INS

R

ER

k2

p3

8

ME

T

CH

K1

Src

AB

L

RS

K1

CH

K2

FY

N

LC

K

PK

Cz

PR

AK

Au

rA

LY

N

MK

2

PK

D2

PK

A

Bis-I 74 95 95 32 55 22 12 -8 6 50 82 16 43 98 78 17 62 78 10 36 43 39 92 72

H-89 91 96 10 36 66 32 3 13 8 11 67 19 28 69 65 -8 24 15 53 61 10 71 93 99

staurosporine 98 100 97 82 99 49 91 73 14 100 100 100 94 99 99 100 100 91 96 100 99 97 99 100

go6976 83 97 95 82 94 68 14 84 59 96 97 93 79 97 85 98 90 18 71 95 94 91 98 98

PP2 12 34 56 29 -5 92 10 15 62 5 20 99 89 29 26 99 97 -14 63 55 98 42 78 96

SB203580 -2 -14 85 28 6 92 -2 6 95 25 5 63 39 11 3 39 53 -21 67 18 61 50 69 78

K252a 86 99 96 98 68 45 70 99 -15 99 100 98 59 99 100 96 97 29 97 100 98 97 99 100

SU6656 -4 19 58 8 10 29 10 -27 -3 47 37 94 72 42 48 96 73 -11 36 94 82 -19 58 70

5iodotubericidin 9 16 74 94 99 95 33 87 25 41 24 42 52 50 45 43 47 20 57 57 54 46 99 76

H-7 2 27 30 -24 8 21 6 -38 18 23 12 2 29 46 9 18 1 -3 60 13 -3 39 86 85

H-9 52 91 93 40 31 42 9 27 42 74 64 10 74 81 52 35 15 65 80 46 13 72 99 94

KN-62 39 82 93 11 89 77 4 55 -2 93 39 76 79 39 58 94 54 13 94 86 80 97 97 97

quercetin 94 81 96 89 97 91 20 98 40 95 9 99 72 94 72 99 52 -374 90 100 98 95 96 100

ProfilerPro: Good Reproducibility of Screening data

ProfilerPro assay workflow is simple

Thaw Assay (enzyme) and Peptide /ATP Plates & Spin

Reconstitute Enzyme In Buffer

Add Compounds To Assay Plate, Mix and pre-incubate @ 28oC for 15 minutes

Add Stop Buffer to Assay PlateMix and spin

Read In Instrument

Add ATP/Peptide solution to Assay Plate

Mix, spin and incubate @ 28oC for 90 min.

• High Throughput– No assay development time– Faster Turnaround time – 1 day vs 2 weeks or more, if outsourced– Direct “in-lab” compound testing

• Sample number– Profile more compounds (up to 12) simultaneously

• Assay Technology– Direct substrate to product ratio measurement with the mobility shift

assay– High quality data– No radioactivity– No antibodies

• Cost– ~1/3 of Out-Sourced Kinase Profiling,

ProfilerPro Kit Review

ProfilerPro Peptide kits ideal for follow-up assays

Benefit of Mobility Shift LabChip Assays

（ 1）直接检测酶促反应的底物和产物，灵敏度高，假阳性和假阴性率低

（ 2）可以覆盖几乎所有的药物筛选酶靶标

（ 3）具有实时动力学检测功能，提高了筛选实验开发和MOA研究的效率，节省了试剂

（ 4）不需要抗体或放射性试剂

（ 5）很高的检测精度，可以检测到其它技术没法检测的弱抑制剂

（ 6）简单快速的开发筛选实验

（ 7）适用于激酶实验中很宽的 ATP浓度范围

（ 8）很小的反应体积（小体积微孔板， 4 － 12ul体系）