Dutch Boltz - Molecular Devices

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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.
Page 1 and 2: Dutch Boltz • Dutch has been at M
Page 3 and 4: Today’s s Presentation • Review
Page 5 and 6: Voltage Dependent FRET Mechanism of
Page 7 and 8: Comparison of the Two “I” Plate
Page 9 and 10: Bleed Down of “FR IPR” Membrane
Page 11 and 12: Components Used To Modify FLIPR to
Page 13 and 14: FRET Dye Protocol • 30,000 HEK-2
Page 15 and 16: 384 Well FLIPR Ratio Traces for a 1
Page 17 and 18: Inhibition of Membrane Depolarizati
Page 19 and 20: Areas for Improvement • Can Optic
Page 21: For More Details and Insight See Ri
Page 25 and 26: Coumarin-Donor Oxonol-Acceptor Fluo

Dutch Boltz - Molecular Devices

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