Shakti BioResearch LLC

Contract Research

& Assay Development Services

1 Bradley Road, Unit 401

Woodbridge, CT 06525

Tel. 203-641-7827

www.shaktibioresearch.com info@shaktibioresearch.com

Who We Are Founded in 2007

5,000 sft of laboratory space

Affiliated with GLP certified Laboratory providing Clinical

Research Service to Pharmaceutical Clients

Equipped with

Biological safety hoods

Incubators

Luminometer

Spectramax plate reader

BioTek ELx405 plate washer,

Thermal Cyclers

Other support instruments

Instruments calibrated regularly to maintain the consistency of

our work

Our Clients

Compound

Profiling

Assay

Development

Stable

Cell Line

Generation

Molecular

Biology

ELISA

Biomarker

Analysis

Protein

Production

Core Competencies

Pre-Clinical R&D

• SiRNA and gain-of-function studies

Target ID and Validation

• Cloning, Mutagenesis

• Stable cell line generation Molecular Biology

• Development of target specific biochemical & cell based assays

• In cell westerns, cell proliferation assay

• Compound profiling

Lead Optimization

• Bacterial, Insect and Mammalian Systems Protein Expression

Translational Medicine

Evaluate effects of kinase inhibitors on signaling pathways in cell lines

Evaluate surface marker antibodies for immuno-histochemicl analysis

Clinical Studies

Clinical PK analysis: Avastin, CRF

Biomarker analysis: VEGF, sVEGFR2, HGF and soluble cMET levels

Clinical Research

BIOMARKER ANALYSIS

ELISA

VEGF ELISA : Standard Curve

Spiked amt

pg/mL

QC

Samples Wells OD R

Conc

pg/mL

MeanConc

pg/mL Std.Dev. CV% Accuracy%

80 QC_Low C8 0.102 65.816 74.023 5.777 7.8 82.3

D8 0.115 76.852 96.1

E8 0.109 71.755 89.7

F8 0.117 78.552 98.2

G8 0.12 81.104 101.4

H8 0.107 70.057 87.6

600 QC_Mid C5 0.724 625.94 619.949 26.63 4.3 104.3

C6 0.69 593.538 98.9

C7 0.742 643.182 107.2

D5 0.688 591.639 98.6

D6 0.756 656.636 109.4

D7 0.706 608.759 101.5

1800 QC_High A5 1.815 1795.76 1864.926 114.397 6.1 99.8

A6 1.888 1884.247 104.7

A7 1.968 1982.895 110.2

B5 1.907 1907.515 106.0

B6 1.71 1670.975 92.8

B7 1.94 1948.166 108.2

VEGF ELISA : Method Validation

All QC samples within range

DEVELOPMENT OF CYTOBLOT ASSAY

Ce

ll L

ine

3

Ce

ll L

ine

1 / S

S

Ce

ll L

ine

4

Ce

ll L

ine

7

Ce

ll L

ine

6

Ce

ll L

ine

5

pAKT

250kD

150kD

100kD

75kD

50kD

37kD

25kD

20kD

15kD

Ce

ll L

ine

1

Ce

ll L

ine

2 / S

S

Ce

ll L

ine

2

Ce

ll L

ine

8 / S

S

Ce

ll L

ine

8

Screening Cell Lines For pAKT Expression

Cytoblot With S473 pAKT Antibodies

LY

-20.0

0.0

20.0

40.0

60.0

80.0

100.0

120.0

-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0

Log Conc.(M)

%In

hib

itio

n

Wortmanin

-20.0

0.0

20.0

40.0

60.0

80.0

100.0

120.0

-9.0 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5

Log Conc.(M)

%In

hib

itio

n

Cytoblot With T308 pAKT Antibodies

LY

-20.0

0.0

20.0

40.0

60.0

80.0

100.0

120.0

-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0

Log Conc.(M)

%In

hib

itio

n

Wortmanin

-20.0

0.0

20.0

40.0

60.0

80.0

100.0

120.0

-9.0 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5

Log Conc.(M)

%In

hib

itio

n

RECOMBINANT PROTEIN PRODUCTION

1. His-tagged protein

2. Thrombin treated His-tagged protein.

~2.75 mg protein digested overnight.

~90% protein got clipped

3. Purified protein. Biotinylated thrombin

removed by streptavidin agarose and

His-tag removed by Ni-NTA resin

Expression Of Recombinant Protein In Bacteria

1 2 3 MW

250kD 150kD

100kD

75kD

50kD

25kD

20kD

15kD

37kD

10kD

1 2 3 MW

250kD 150kD

100kD

75kD

50kD

25kD

20kD

15kD

37kD

10kD

4 - 12% SDS/PAGE

250kD

150kD

100kD

75kD

50kD

37kD

25kD

20kD

15kD

10kD

1 2 3 4

1. Control protein

2. Conditioned Media

3. Clone 65-18 Sup

4. Clone 65-18 Sup from

suspension culture

Precursor protein

Processed alpha subunit

Processed beta subunit

Expression Of Recombinant Protein In CHO Cells

COMPOUND PROFILING

Cell Titer Glo Assay (CTG)

Cell Titer 96 (MTS)

Parameter measured ATP MTS reduction

Incubation Time 10 minutes 1 - 4 hours

Sensitivity 50 cells 800 cells

Sample Type Suspension or adherent cells

Suspension or adherent cells

Detection Luminescence Colorimetric

Cell Proliferation Assays

CTG and MTS based

-20.0

0.0

20.0

40.0

60.0

80.0

100.0

120.0

-9.5 -9.0 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5

Log Conc.(M)

%In

hib

itio

n

IC50= 19 nM

Cell Proliferation Assay CTG Assay

• Eight point dose-response curve in duplicates

• Compounds dosed for 72 hours

Luminescence Conc. (mM)

35756 40383 1

41029 44479 0.33

39864 40812 0.11

64838 66503 0.04

136117 139179 0.012

159310 166269 0.004

177348 168209 0.001

179366 173555 DMSO

Cell Proliferation Assay MTS Assay

• Eight point dose-response curve in duplicates

• Compounds dosed for 72 hours

• Absorbance read at 490 nm

-20.0

0.0

20.0

40.0

60.0

80.0

100.0

120.0

-8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5

Log Conc.(M)

%In

hib

itio

n

IC50= 342 nM

A490nm Conc (mM)

0.91 0.85 10

0.96 0.94 3.33

1.23 1.26 1.11

2.05 2.05 0.37

2.80 2.72 0.12

2.87 2.80 0.04

2.77 2.80 0.014

2.87 2.77 DMSO

Cell lines

Compound 1

IC50 (nM)

Compound 2

IC50 (nM) Mutation

HT29 20 21 BRAF

Colo205 20 17 BRAF

HepG2 17 15 N-Ras

HCT116 461 544 K-Ras

Caki1 > 1000 > 1000 WT

Proliferation Assay in Mutant Cell Lines

Mechanism of Action Assay Effect on ERK Signaling Pathway

Phospho - ERK1/2

Phospho - ERK1/2

Total - ERK1/2

Total - ERK1/2

Compound 1

Compound 2

DM

SO

0.0

37

m M

0.1

1 m

M

0.3

3 m

M

1.0

m M

STABLE CELL LINE GENERATION

TARGET VALIDATION

Ce

ll L

ine

1

Ce

ll L

ine

2

Ce

ll L

ine

3

Ce

ll L

ine

4

Ce

ll L

ine

5

Protein1

Protein 2

Protein 3

Protein Expression Profile In Cancer Cell Lines

b-actin

• Different cell lines were screened for the expression of proteins of interest

• Cell Line 1 and 4 expressed all three proteins of interest and hence chosen for siRNA studies

• Cell Line 1 had better transfectability

SiRNA Transfections In Cancer Cell Line 1

• Proliferation measured after 67 hours of transfection

• Two validated SiRNAs/ gene or four not-validated SiRNAs were used

• Gene A SiRNAs #5 and #6 had no effect on proliferation

• Gene B SiRNA #2 had significant effect on proliferation

• Gene C SiRNA #6 reduced proliferation by ~30%

• Gene D SiRNA #5 reduced proliferation by ~40%

0

50000

100000

150000

200000

250000

300000

350000

400000

No tr

eatm

ent

No R

NA

+ve -ve

Gen

eA 5

Gen

eA 6

Gen

eB 1

Gen

eB 2

Gen

eB 4

Gen

eB 5

Gen

eC 5

Gen

eC 6

Gen

eD 5

Gen

eD 6

Gene A Gene B Gene C Gene D

250kD

150kD

100kD

75kD

50kD

37kD

20kD

25kD

No T

rea

tme

nt

Mo

ck T

ran

sfe

ction

+ve

SiR

NA

co

ntr

ol

-ve

SiR

NA

co

ntr

ol

Ge

ne

D S

iRN

A #

5

Ge

ne

D S

iRN

A #

6

ProD

Actin

• Protein extracts were made 48

hrs post-transfection

• GeneD SiRNA #5 showed ~50%

inhibition in proliferation assay, and

reduced protein levels

• GeneD SiRNA #6 did not show

any inhibition in proliferation assay,

nor did it reduce protein levels

significantly

• Blot was reprobed with actin

antibodies (CST # 4967)

• Proliferation assay was for 72 hrs

Reduction Of Target

Protein In SiRNA

Transfected Cells

!

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