Dutch Boltz - Molecular Devices

Dutch Boltz
• Dutch has been at Merck 25 +years. He Has developed
the Cell Analysis Facility at Rahway into a company
wide resource for fluorescence cell based applications.
He got his first FLIPR in 1996. Also, the same year, his
laboratory began a long time collaboration with
Cellomics developing ArrayScan and the nuclear
translocation assay as well Cellomics Store. Today, he
will present “A A Simple Dual-Laser Configuration for
Screening Ion Channels on FLIPR Using Ratiometric
Coumarin-DiSBAC
2 FRET Voltage Sensor Probes”
6/14/2004 Cardiovascular Diseases 1

A Simple Dual-Laser Configuration for
Screening Ion Channels on FLIPR Using
Ratiometric Coumarin-DiSBAC
2 FRET Voltage
Sensor Probes
Richard F.Wnek, Randal M. Bugianesi, Paula M. Dulski, Anna Sirotina
ina-
Meisher, Joe Jackson, Dutch Boltz
Departments of CardioVascular Diseases and Ion Channels, Merck Research R
Laboratories & Product Development, Molecular Devices
6/14/2004 Cardiovascular Diseases 2

Today’s s Presentation
• Review of FRET Membrane Potential Dyes
• Why FRET on FLIPR?
• Design of 2 Ion Plate Readers
• Differences and Reasons
• FLIPR FRET Modification
• Proof of Concept
• Data Comparison 150 Compounds Medicinal Chemistry
• Possible Improvements
• Conclusions
• Rich Wnek’s Poster
6/14/2004 Cardiovascular Diseases 3

Why a FRET Membrane Potential Dye?
• Provides a Ratiometric Measurement
• Cell Number Insensitive
• Temperature insensitive
• Instantaneous Membrane Potential Sensitivity
Measurement
• Increased Signal to Noise – Donor/FRET Only
• Time Domain of Single Channels
6/14/2004 Cardiovascular Diseases 4

Voltage Dependent FRET Mechanism
of Voltage Sensor Probes
+ + + - - -
V m
+ + +
- - -
-70mV
+50mV
+ + +
Extracellular
FRET
V m
---
Extracellular
Intracellular
---
Donor CC2-
DMPE
Acceptor
DiSBAC 2
Intracellular
+ + +
6/14/2004 Cardiovascular Diseases 5

Why Combine FLIPR and FRET Dye?
• Utilize Optimal Parts of Two Approaches
• Increased Sensitivity? on FLIPR
• Increased Reliability?
• Increased Reproducibility ?
• Greater Throughput for Longer Time Courses
> 1-21
2 minutes
• Standardization between Screening Platforms
• Increase Throughput- Multiple Instrument Campaign
6/14/2004 Cardiovascular Diseases 6

Comparison of the Two “I” Plate Readers
“IPR”s
Micro titer
Well
PMT
PMT
2 FL
Slider
Micro titer
Well
Cooled
CCD
6/14/2004 Cardiovascular Diseases 7

IPR Design Criteria
FLIPR
• Ion Channel Screening
• Membrane Potential Dye
• High Throughput
• Kinetic Measurement
• Simultaneous
• Whole Plate
• Temperature Control
• Biology
• Fluorescent Signal
• 488 nm Excitation
“FR-IPR”
• Ion Channel Screening
• More Sensitive S/N
• PMT
• More Rapid Kinetics
• < 20 msec
• Partial Plate PMT
• “Membrane/Potential”
FRET Pair
• Multi Wavelength
(409nm)
6/14/2004 Cardiovascular Diseases 8

Bleed Down of “FR IPR” Membrane
Potential Signal Over Time
16 Well Addition/Detection Array
Negative Controls
Well Acquisition Time
Order
6/14/2004 Cardiovascular Diseases 9

Dual Laser Optical Modification of FLIPR 384
6/14/2004 Cardiovascular Diseases 10

Components Used To Modify FLIPR to
Utilize the Membrane Potential FRET Dyes
• Coherent Innova 302C Laser
• Vintage Newport Research Optical
Positioning Components
• CVI Laser Corp Special Order Optics
• Tunable Laser Mirror 400 nm center 45deg
polarization preserving TLM1-400
400-45p-2025
2.000 x 0.25” 100nm
• Laser Window Plain Window Fused Silica W1-
PW- UV-409
409-488-4545
• Long Wave Pass Dichroic Beam splitter LWP-
45-R400
R400-T480-PW-2025
Dutch 2025-UV & Beam Steering Components
• 460/40
Circa 1980
• 565/50
6/14/2004 Cardiovascular Diseases 11

Comparison of Ratio metric Data Acquisition
on the Two “IPR”s
Micro titer
Well
PMT
PMT
2 FL
Slider
Micro titer
Well
Cooled
CCD
6/14/2004 Cardiovascular Diseases 12

FRET Dye Protocol
• 30,000 HEK-2 cells/well
• Incubate overnight.
• Stain Plates with 50µL of 10µM DiSBAC2
• Wash once with 50µL of PBS,
• Stain with 30µL of 5µM CC2-DMPE
• 50µL saline wash.
• Initiate by a 25µL of 30µM Deltamethrin/100nM Brevatoxin
(PbTX-3).
• Experiment is performed at 25°C on both Instruments
• Data Exported to Excel
• Ratio Calculated & Displayed with Original Data in Excel
6/14/2004 Cardiovascular Diseases 13

FLIPR Eliminates Bleed Down of FRET Signal
6/14/2004 Cardiovascular Diseases 14

384 Well FLIPR Ratio Traces for a 16
Point Titration of Four Compounds
Compound
A
Compound
C
Compound
B
Compound
D
6/14/2004 Cardiovascular Diseases 15

Inhibition of Membrane Depolarization by
Compounds A and B
Compound A
IC 50 = 0.074µM
IC 50 = 0.066µM
Compound B
IC 50 = 0.35µM
IC 50 = 0.74µM
FLIPR
FR IPR
6/14/2004 Cardiovascular Diseases 16

Inhibition of Membrane Depolarization by
Compounds C and D
Compound C
IC 50 = 0.13µM
IC 50 = 0.31µM
Compound D
IC 50 = 0.17µM
IC 50 = 0.45µM
FLIPR
FR IPR
6/14/2004 Cardiovascular Diseases 17

Correlation of 150 Medicinal Chemistry
Compounds at 1µM 1 M or 3µM on the
Modified FLIPR and “FR IPR”
6/14/2004 Cardiovascular Diseases 18

Areas for Improvement
• Can Optics Be Better?
• Not a True simultaneous Ratio
• Faster Integration Times
• Faster Filter Changing
• Redisplay of The Two Color Individual
Traces
• Ratio metric Calculation and Display
6/14/2004 Cardiovascular Diseases 19

Conclusion of 488 nm/409 nm FLIPR FRET
Membrane Potential Application Study
• An Additional Laser Beam Can be Combined into The FLIPR 384 Optics
• Original Operation “Unaffected”
• Coumarin DiSBAC2 FRET Data Can Be Collected
• FLIPR Eliminates Signal Bleed Down
• Excellent Titration Data Correlation Between Plate Readers Tested
• IC 50s Consistently Lower
• The System is Robust and Yields Reproducible Data
• Kinetics Appeared to be Faster Than Molecular Devices Red Dye > 2
Minutes
6/14/2004 Cardiovascular Diseases 20

For More Details and Insight See
Rich Wnek’s Poster
6/14/2004 Cardiovascular Diseases 21

IPR”s s and Their Niche
FLIPR
• > 5-105
sec Time course
• Plate Read = 1 Time
course
• 5 Sec = 5 sec
• 1 minute plate exchange
• Calcium
• Temperature Control
“FRIPR”
• < 1-51
5 sec Time course
• Heterogeneity?
• Plate Read > 12 – 24x
Time course
• 5 sec = > 60 – 120 sec
• Calcium
• No Temperature Control
6/14/2004 Cardiovascular Diseases 22

Abstract
Ion channels are an attractive class of drug targets because of their involvement in a diverse range of
cardiovascular, inflammatory, and neurological disorders. Their pharmacological complexity proves
advantageous for developing highly selective drugs that affect only specific functional states of the channel.
Advancements in fluorescent dyes and optical detection technologies have provided drug discovery programs
with functional screening assays capable of detecting ion channel modulation in an HTS format. We have
developed a membrane potential kinetics screen for a FLIPR-384 instrument that utilizes a FRET-based
coumarin-DiSBAC 2
dye system to screen inhibitors of ion channel X expressed in HEK293 cells. We have
modified a FLIPR-384 instrument with a 409nm krypton laser, introducing the beam co-linear to the 488nm
argon laser path by additional beam steering optics in order to overcome limitations which previously
restricted the use of such FRET based dyes on FLIPR . A number of channel X specific compounds were
screened on the modified FLIPR-384 and compared to results generated on the standard kinetic plate reader.
There was good correlation between both systems for all tested compounds. The modified FLIPR had a
slightly increased sensitivity and a 12-24 factor increase in throughput. Running this assay in ratiometric
mode on FLIPR, reading all 384 wells simultaneously, eliminates the run-down of signal and variability
observed on the other plate reader. These findings demonstrate the feasibility of using a modified FLIPR-384
as an HTS platform utilizing FRET based voltage sensors to identify novel ion channel modulators.
Additionally, the same configuration with an argon-krypton laser substitution for the argon primary laser
would permit the use of virtually any dye on FLIPR with the possibility of simultaneously following two
distinct physiological sensors .
Introduction
Historically, ion channel HTS assays have taken the form of membrane potential or calcium flux population
kinetics. More recent approaches have shifted toward the use of membrane potential sensitive fluorophores
that undergo redistribution within the plasma membrane in response to voltage changes. One such approach
makes use of DiSBAC 4
, or a similar single excitation, single emission dye to measure time courses greater than
10 seconds. A more rapid technique was later developed that utilizes a Coumarin-DiSBAC 2
FRET dye pair to
deal with time courses less than 10 seconds. It has the distinct advantage of functioning as a ratiometric dye
with increased temporal resolution, reproducibility and throughput; advancing measurements beyond the
detection of steady-state changes in membrane potential and avoiding the need for pharmacological
6/14/2004 Cardiovascular Diseases 23
modification of rapidly inactivated or desensitized ion channels.

A Dual-Laser Configuration on a FLIPR 384Instrument
In order to utilize the FRET based voltage sensor probes for ion channel screening on FLIPR a
409nm krypton laser was introduced onto the instrument. At left, the krypton-argon laser
substitution diagram for the primary argon laser. The 409nm beam was introduced co-linear to
the 488nm argon laser path by the addition of beam steering optics. Two dichroic filters are
required to pass the krypton light beam through a mirror and into the FLIPR optical detection
path. The argon light beam passes through the dichroic filter and mirror into the FLIPR optical
detection path. At right, a photograph of the 409nm krypton laser beam path through the beam
steering optics and into the FLIPR. The substitution of the primary light source in conjunction
with the use of multiple filter sets allows one to use a wide range of distinct fluorescent probes.
6/14/2004 Cardiovascular Diseases 24

Coumarin-Donor
Oxonol-Acceptor
Fluorescent Dye Components of the FRET Membrane Potential Assay
A two dye configuration is used in the FRET based membrane potential assay. At left, the
coumarin labeled phospholipid CC2-DMPE molecule which partitions itself into the outer
leaflet of the plasma membrane and serves as the FRET donor. Negative charges from the
coumarin and phosphate groups prevent the CC2-DMPE donor molecule from crossing the
phospholipid bilayer. At right, the negatively charged bis-(1,3-dialkylthiobarbituric acid)
trimethane oxonol acceptor molecule whose position within the bilayer is determined by the
membrane potential of the cell. Both components are very bright fluorophores with a large
spectral separation between emissions making signal separation and detection feasible.
Coumarin excitation is at 410nm and emission at 460nm as compared to Oxonol with an
excitation at 540nm and emission at 570nm.
6/14/2004 Cardiovascular Diseases 25

Conclusions
The addition of a 409nm krypton laser onto a FLIPR-384 instrument has provided our
laboratory with the means to utilize FRET based voltage sensor probe technologies as a
screening tool for identifying ion channel modulators in an HTS format. As an initial study
we developed a membrane potential kinetics screen for the modified FLIPR that utilizes a
FRET-based coumarin-DiSBAC 2 dye system to screen inhibitors of ion channel X expressed
in HEK293 cells. Results from the study indicate that the percent inhibition of
depolarization for channel X compounds generated from data collected on the modified
FLIPR-384 instrument correlates well with that obtained from the standard kinetic plate
reader. The modified FLIPR, in addition, had comparable sensitivity and a 12-24 factor
increase in throughput. The ability to run the instrument in ratiometric mode in conjunction
to imaging all 384 wells simultaneously eliminates concerns such as variability and run down
of signal over time which was observed on the other plate reader during channel X
screening. These findings demonstrate the effectiveness of the modified FLIPR as an
alternative HTS platform for FRET based ion channel screening. In addition, we could use
virtually any dye on the modified FLIPR with the possibility of simultaneously following two
distinct physiological sensors.
References
Gonzalez, J.E. and Maher M.P. Receptor and Channels, 8: 283-295 (2002).
Mattheakis L., Savchenko, A. Current Opinions in Drug Discovery and Development, 4(1): 124-134 (2001).
Gonzalez, J.E., Oades K., Leychkis, Y., Harootunian, A., Negulescu, P.A. Drug Discovery Today, 4(9):
431-439 (1999).
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