new fluorescent technology for protease activity detection€¦ · new fluorescent technology for...

New Fluorescent Technology for Protease Activity Detection

Diana L. Hulboy, Ph.D.

VP, Marketing and Business Development

Enzium, Inc.

Proteases

*The three detection approaches are complementary.

Disease Targets Organisms Industries Detection*

Biomarkers Mammalian Pharma Mass Spec

Diagnostics Microbial Diagnostics Antibodies

Drug Discovery Viral Basic Research Enzyme Activity

Veterinary

Foodcontamination, Bioremediation

Cosmetics (Ageing)

Traditional Fluorescent Based Protease Activity Detection Technologies

• These technologies use short peptides encoding the core protease recognition sequence.

• They require attachment of a moiety at optimum distance from the attached fluorophore: quencher for FRET/TRF; fluorescence lifetime modulator for FLT.

• Cleavage by the target protease results in removal of the moiety from the peptide, giving the measured signal emitted by the fluorophore.

FRET, TR-FRET,Q-FRET

TRF FLT

Fluorescence Resonance Energy Transfer

Time ResolvedFluorescence

Fluorescence Lifetime

New EnSens® Protease Activity Detection Technology Is the Alternative to FRET/TRF/FLT

Protease Sample+

EnSens® : Dye Mix

Start to Finish: ~30 – 60 minutes

EnSens® Substrate +

Far Red Dye

In Solution

Quantitative/QualitativeFluorescence Output

Indicates Protease Activity

1. Mix Kit Components 2. Add Sample 3. Read Signal

Dye

ProteaseRecognitionSequence

Protease Detection Kit Add Sample Collect Data

Active Protease In Sample

Activated Dye

EnSens® vs. FRET/TRF/FLT

FRET/TRF/FLT EnSens®

Substrates are short; not very selective or physiologically relevant

Substrate can incorporate full recognition site sequence, yielding

more relevant, high-quality hits

Design limits variety of substrates, requires experimentation to

develop new substrates

Substrate is modular, allowing easy swap of protease recognition site —> substrate library screening

Limited stability in vitro and in vivo Substrate is an scFv-based protein, which confers high stability in vitro

and in vivo

Substrate Fluorophore Applications Data Conditions✓



Fluorophores can be photobleached more easily than

EnSens fluorophore

EnSens se-Red fluorophore is resistant to photobleaching up to

48 hours

Many fluorophores have shorter emission wavelengths, more

subject to interference

Long emission wavelength (665nm) reduces interference from other assay components

Substrate and fluorophore are one entity—once quenched, it’s done

Substrate and fluorophore are separate, so the fluorophore is constantly renewed in solution

Free fluorophore can mask energy transfer (self-quenching)

No energy transfer involved, minimum self-quenching




Not easily applicable to cellular or in vivo applications

Established protocols for flow cytometry, standard and 3D cell

culture microscopy

Can involve complicated/expensive equipment

Standard plate or cuvette fluorimeter is sufficient

Can involve complicated algorithms and data manipulation

Quickly and directly convert RFU readouts to reaction velocity, kcat,

Km, IC50, etc.

Higher background than EnSens High signal-to-noise ratios: 6-20X

Substrate Fluorophore Applications Data Conditions✓ ✓



Can require higher enzyme and substrate concentrations

Lower enzyme and substrate concentrations mean more competitive inhibitor hits

Can be pH sensitive Stable in wide range of pH, salt, & solvent conditions

Low aqueous solubility Substrate and dye are highly soluble


EnSens® Assays are Repeatable, Reproducible, and Robust

Each Assay Run During Validation was:

• Repeatable - Run in Triplicate with little variance • Reproducible - Each Triplicate Run on 3 different days

by different Research Associates -• Robust - Scaled from 100 uL down to 10 uL in varying

conditions

Validation Data Provided By Dr. David Schultz at The Core Molecular Screening Facility at the Wistar Institute of Philadelphia.

3 R’s Signal : Noise Selective Novel Quantitative Versatile✓

EnSens® Gives Repeatable and Reproducible Kinetic Activity Curves from Reagent to Reagent



0

5000

10000

15000

5 15 25 35 45 55 65 75 8562

0/6

60

nm

Flu

ore

sce

nce

Em

issi

on

(R

FU

Time (Min)

EnSens Caspase-3 Substrate

100ng Caspase-3

50ng Caspase-3

25ng Caspase-3

0ng Caspase-3 0

10000

20000

30000

40000

4 12 20 28 36 44 52 60 68 76 84

62

0/6

60

Flu

ore

sce

nce

Em

issi

on

(R

FU)

Time (Min)

EnSens Thrombin Substrate8ng thrombin

4ng thrombin

2ng thrombin

1ng thrombin

0.5ng thrombin

no enzyme

0

10000

20000

30000

40000

50000

4 12 20 28 36 44 52 60 68 76 84

62

0/6

60

nm

Em

issi

on

Fl

uo

resc

en

ce (

RFU

)

Time (Min)

EnSens MMP-14 Substrate

100ng MMP-14

50ng MMP-14

25ng MMP-14

12.5ng MMP-14

no enzyme

0

500000

1000000

1500000

2000000

2500000

3000000

0 12 24 36 48 60 72 84 96 108120

62

0/6

60

nm

Em

issi

on

Flu

ore

sce

nce

(R

FU)

Time(Min)


10ng MMP-2

5ng MMP-2

2.5ng MMP-2

1.25ng MMP-2

0.62ng MMP-2

0.31ng MMP-2

0.16ng MMP-2

No MMP-2

EnSens® Is Stable in Varying Conditions Often Used in Biological Sample and Enzyme Kinetic Assays

Signal Over pH Change Signal Over NaCl Change

Signal Over Increase in DTT Signal Over Increase in EDTA

Validation Data Provided By Dr. Matthew Farber at University of the Sciences of Philadelphia, Department of Biology.

Rel

ativ

e Fl

uo

resc

ent

Sign

al In

ten

sity


EnSens® Produces Higher Signal-to-Noise Ratios Than FRET



Protease Target EnSens® FRET

Caspase 3 27 X 12 X

MMP-2 12 X 15 X

MMP-14 14 X 3 X

MMP-25 18 X None Available

Thrombin 12 X 10 X

Result: Fewer false positives and negatives

EnSens® Reagents Display Superior Selectivity

Validation Data Provided By Dr. Jeffrey Smith at Center for Proteolytic Pathways at the Sanford-Burnham Medical Research Institute.


0

20000

40000

60000

80000

100000

120000

MMP-2 MMP-9 MMP-14 MMP-15 MMP-16 MMP-17 MMP-24 MMP-25

kcat

/Km

EnSens MMP-2 Substrate Selectivity

✓

EnSens® Substrates Display Superior Selectivity



0

500

1000

1500

2000

2500

3000

3500

4000

0

10

20

30

40

50

60

70

80

90

10

0

11

0

12

0

Flu

ore

scen

ce O

utp

ut

(RFU

)Time (Min)

EnSens MMP-14 Substrate Selectivity

MMP-14

MMP-9

MMP-7

MMP-2

MMP-3

No MMP

0

280000

560000

840000

1120000

1400000

1680000

1960000

0 10 20 30 40 50 60Flu

ore

scen

ce O

utp

ut

(RFU

)

Minutes


MMP-14

MMP-2

No MMP

0

2000000

4000000

6000000

8000000

10000000

12000000

14000000

0 10 20 30 40 50 60

Flu

ore

scen

ce O

utp

ut

(RFU

)

Minutes

FRET ‘Selective’ MMP-14 Substrate

MMP-14

MMP-2

No MMP

Develop Novel, Selective Substrates with EnSens®

Validation Data Provided By Dr. Jeffrey Smith at Center for Proteolytic Pathways at the Sanford-Burnham Medical Research Institute.


0

20000

40000

60000

80000

100000

120000

140000

MMP 25 MMP 17 MMP 9 MMP 8

EnSens MMP-17/25 Substrate Challenged with Related MMP Proteases

Kca

t/km

*Note: MMP17 and MMP25 protease are biologic mimics that recognize the same substrates

✓ ✓

y = -11576x2 + 36973x + 5861.4R² = 0.98691

0

10000

20000

30000

40000

0 0.5 1 1.5 2 2.5

Max

imu

m R

FU/m

in

Thrombin Protease (ng)

Thrombin Standard Curve

EnSens® Assays Provide Direct Output for Easy and Accurate Quantitation of Active Protease At Low

Concentrations When Compared to Standard Curves


Actual Thrombin in Well

EnSens Reading

Unknown A 1.00ng 0.941ng

Unknown B 0.125ng 0.133ng

Unknown C 0.250ng 0.232ng

Thrombin Standard Curve was built by measuring the protease activities of known thrombin amounts (based on protein concentration) (Fig A).

In this double-blinded study, the activities of three unknown amounts of thrombin were measured, and their thrombin protein amounts extrapolated from the standard curve (Fig B).


A.

B.

EnSens® Gives IC50 Curves Comparable to FRET in High Throughput Protease Inhibitor Screens

IC50s (uM) Thrombin Inhibitor 1

Thrombin Inhibitor 2



Inhibitor Dabigatrin

EnSens 0.039 0.048 0.593 1.347 0.002

FRET 0.044 0.038 0.635 1.677 0.001


FRET IC50 CurveEnSens IC50 Curve


Endogenous MMP-25 Protease Activity

Plasmids Encoding EnSens® Can Be Transfected Into Cell Lines and Expressed as Cell Surface Protease Activity

Detection Labels for High Content Screening

Exogenous HRV 3C Protease Activity

Data provided by Dr. Alex Strongin Lab at the Sanford-Burnham Medical Research Institute; and by Dr. John Hollaren at the Molecular BioSensor and Imaging Center Carnegie Mellon University.

Controls: Cell Auto-Fluorescence

EnSens Detects MMP-25 ProteaseActivation in MCF-7Breast Cancer Cells


Before Protease Addition

GFP EnSens GFP EnSens

After Protease Addition

3-D Imaging of Breast Cancer Cell Migration:Tracking MMP-14 Activity Using EnSens®

Data provided by Jennifer Rothberg in the Bonnie Sloan Lab at Wayne State University Department of Pharmacology.

MMP-14 ProteaseExpressing Cells

MMP-14 NegativeCells

Wit

ho

ut

MM

P In

hib

ito

rW

ith

MM

P In

hib

ito

r

EnSens® MMP-14 Substrate Embedded In 3D Gel Matrix is Used to Track MMP-14 Activation During Cancer Cell Migration

Red: CellTrackerTM OrangeWhite: MMP-14 Activity

Note: MMP inhibitor unable to diffuse to center of cell mass


EnSens® Reagents Can Be Easily Attached to Fluorescent Beads for Flow Cytometry Based Multiplex Assays

UnCleavable and Cleavable

Thrombin EnSens Substrates

Conjugated to Beads Are Mixed

And Treated With Thrombin

Protease

P3P2

P3

P2

Be

ad S

ign

alEn

Sen

s Si

gnal

Data provided by Dr. Jody Franke in the Molecular BioSensor and Imaging Center, Carnegie Mellon University

+

+ Thrombin

P2

P2

+

P3

P3

+


• Enzyme Activity Detection Is a Third Method of Detection• Complements MS and Antibody Methods Across the Entire

R&D Pipeline• Enzium EnSens® Technology Is a Superior New Method

Research Drug Screen

Cell TestingCompanion

Testing

MSELISA

MSELISA

MSELISA

MSELISA

Pip

elin

eP

has

e

• Fluorescent Microscopy

• Fluorimetry• Cytometry• Cell Assays• Bead Assays

• HTS• HCS• Flow

Cytometry

• HCS• Fluor.

Imaging• Tissue /Cell

Analysis

• Patient Sample Analysis

• Multiplexing

EnSe

ns

Ap

plic

atio

ns

Enzium’s Current Product Offering

For 96 or 384 well plates

Protease Target Kit Name Catalog Number

Available Now -- Custom Substrate Service

Coming Soon – Substrate Library Screening

MMP-2 EnSens® MMP-2 Activity Detection Kit 032014-01




ADAM10 EnSens® ADAM10 Activity Detection Kit 032014-08

ADAM17 (TACE) EnSens® ADAM17 Activity Detection Kit 032014-09

Furin EnSens® Furin Activity Detection Kit 032014-05

Thrombin EnSens® Thrombin Activity Detection Kit 032014-06

Factor Xa EnSens® Factor Xa Activity Detection Kit 032014-07

Enzyme Activity Detection Made EasyMix. Read. Innovate.www.enziumlabs.comMix. Read. Innovate.

new fluorescent technology for protease activity detection€¦ · new fluorescent technology for...

Documents