![Page 1: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/1.jpg)
New Fluorescent Technology for Protease Activity Detection
Diana L. Hulboy, Ph.D.
VP, Marketing and Business Development
Enzium, Inc.
![Page 2: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/2.jpg)
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)
![Page 3: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/3.jpg)
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
![Page 4: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/4.jpg)
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
![Page 5: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/5.jpg)
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✓
![Page 6: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/6.jpg)
EnSens® vs. FRET/TRF/FLT
FRET/TRF/FLT EnSens®
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
Substrate Fluorophore Applications Data Conditions✓
![Page 7: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/7.jpg)
EnSens® vs. FRET/TRF/FLT
FRET/TRF/FLT EnSens®
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✓ ✓
![Page 8: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/8.jpg)
EnSens® vs. FRET/TRF/FLT
FRET/TRF/FLT EnSens®
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
Substrate Fluorophore Applications Data Conditions✓
![Page 9: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/9.jpg)
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✓
![Page 10: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/10.jpg)
EnSens® Gives Repeatable and Reproducible Kinetic Activity Curves from Reagent to Reagent
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✓
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)
EnSens MMP-2 Substrate
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
![Page 11: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/11.jpg)
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
3 R’s Signal : Noise Selective Novel Quantitative Versatile✓
![Page 12: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/12.jpg)
EnSens® Produces Higher Signal-to-Noise Ratios Than FRET
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✓
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
![Page 13: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/13.jpg)
EnSens® Reagents Display Superior Selectivity
Validation Data Provided By Dr. Jeffrey Smith at Center for Proteolytic Pathways at the Sanford-Burnham Medical Research Institute.
3 R’s Signal : Noise Selective Novel Quantitative Versatile✓
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
✓
![Page 14: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/14.jpg)
EnSens® Substrates Display Superior Selectivity
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✓
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
EnSens MMP-14 Substrate
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
![Page 15: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/15.jpg)
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.
3 R’s Signal : Noise Selective Novel Quantitative Versatile✓
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
✓ ✓
![Page 16: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/16.jpg)
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
Validation Data Provided By Dr. David Schultz at The Core Molecular Screening Facility at the Wistar Institute of Philadelphia.
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).
3 R’s Signal : Noise Selective Novel Quantitative Versatile✓
A.
B.
![Page 17: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/17.jpg)
EnSens® Gives IC50 Curves Comparable to FRET in High Throughput Protease Inhibitor Screens
IC50s (uM) Thrombin Inhibitor 1
Thrombin Inhibitor 2
Thrombin Inhibitor 3
Thrombin Inhibitor 4
Inhibitor Dabigatrin
EnSens 0.039 0.048 0.593 1.347 0.002
FRET 0.044 0.038 0.635 1.677 0.001
Validation Data Provided By Dr. David Schultz at The Core Molecular Screening Facility at the Wistar Institute of Philadelphia.
FRET IC50 CurveEnSens IC50 Curve
3 R’s Signal : Noise Selective Novel Quantitative Versatile✓
![Page 18: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/18.jpg)
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
3 R’s Signal : Noise Selective Novel Quantitative Versatile✓
Before Protease Addition
GFP EnSens GFP EnSens
After Protease Addition
![Page 19: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/19.jpg)
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
3 R’s Signal : Noise Selective Novel Quantitative Versatile✓
![Page 20: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/20.jpg)
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
+
3 R’s Signal : Noise Selective Novel Quantitative Versatile✓
![Page 21: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/21.jpg)
• 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
![Page 22: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/22.jpg)
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
MMP-9 EnSens® MMP-9 Activity Detection Kit 032014-02
MMP-14 EnSens® MMP-14 Activity Detection Kit 032014-03
MMP-25 EnSens® MMP-25 Activity Detection Kit 032014-04
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
![Page 23: New Fluorescent Technology for Protease Activity Detection€¦ · New Fluorescent Technology for Protease Activity Detection Diana L. Hulboy, Ph.D. VP, Marketing and Business Development](https://reader033.vdocuments.us/reader033/viewer/2022042309/5ed5938366e01e16003ba1d9/html5/thumbnails/23.jpg)
Enzyme Activity Detection Made EasyMix. Read. Innovate.www.enziumlabs.comMix. Read. Innovate.