⚐ Realizing the potential of IonWorks screening in a drug discovery ...

Realizing the potential of IonWorksscreening in a drug discoveryenvironmentBenjamin WilenkinIon Channel GroupCNS BiologyPfizer GrotonMolecular Devices User MeetingJanuary 2008

Outline• Ion channel drug discovery at Pfizer– Brief description of resources and team• Infrastructure– Hardware investment– IonWorks applications in a drug discovery environment• Case Study– Na v Assay• Challenges/Issues

Groton ion channel drug discoveryCNS ion channel lab groupsGrotonAnn Arbor• Re-deployment from AA to Groton• Hardware upgrade to 48 channeldispensing head and fluidics25 lab members

New technologies facilitate an integratedapproach to ion channel drug discoveryIdea Lead development Candidate drugThroughput/capacity1000’s 100’s 10’s 1’sIonWorks(384)QPatch(48)Manual(1 – serial)FLIPR(384)OpusXpress(8)

Electrophysiology has an integralrole in iterative ion channel drug designDesignSYNTHESISFLIPRSingle pointIon channelmodulatorChemical spaceexpansionHT EPHYSElectrophysiologyIC 50 / use dependencyFLIPRretestIC 50• Functional ion channel screeningnot rate limiting• Unique opportunity in ion channeldrug discovery• HTS electrophysiology provides anideal tool to discern detailed SARDrugproperties

IonWorks application in drug discoverycase study•Na v Assay– Cell husbandry and preparation– Assay format– Indices of efficacy– Targeting pathophysiological states– Facilitate lead compound selection

Assessment of cell growth parameters areparamount to maximize screening success• IncuCyte – networked, incubator-based cell imaginganalysis system– Eliminates subjectivity– Characterizes growth kinetics– Characterizes most appropriate seeding densities– Evaluate expression as a function of confluence– Key for consistency and experimental scheduling

Cell passage and growth phase are an integralpart of assay designNa v Cell Confluence (%) vs Time (hrs)Passage 7150K cells/ml50K cells/mlPassage 17Seeding densities influence cell growth ratesGrowth rates change over time withincreasing cell passage3 hours – 10% Confluence 48 hours – 40% Confluence 72 hours – 70% Confluence

Growth kinetics dictate experimental timingThe optimal assay window is defined by desired growth kinetics andexperimental timing

Cell preparation affects seal quality and currentamplitudeAverage Seal Resistance350~ 180 MΩ ~ 268 MΩSeal Resistance (MOhms)300250200150100Versene PrepIonWorks HT Experimental RunsVersene / Trypsin PrepAverage seal resistance is increased when a50:50 trypsin/versene mix is used to lift the cellscompared to that of versene aloneAverage current amplitude is increased when a50:50 AmphoB/Nystatin mix is used to as theperforating agent relative to just AmphoB

Variables influencing assay success• Cell husbandry and preparation– Cell Passage– Cell Density– Trypsin/versene cocktail to lift cells– Slow spin prior to final suspension– Gentle trituration to break-up pellet prior toexperiment start* * * Gentle Gentle Gentle * * *• Compound preparation– Glass vials– Low-binding compound plates– All dilutions made in DMSO

Assay format• Single hole HT mode• Pulse to -10mV from -55mV 25times @ 10Hz• First run of day from -100mV– Keep track of relative current– Provides estimate of voltagecontrol• 6 compounds/plate• Full 7-pt IC 50 ’s• One row vehicle control• Four plates/day – 24cmpds/day• 96 cmpds/week

The importance of voltage control onIonWorksNa v Inactivation~ 11mVThe steady-state inactivation curve indicates that an 11mV shift involtage can yield a 50% shift in current amplitude.

Voltage-dependence of compound xpharmacologyCompound x IC 50vs. Holding Potential80706050IC 50(µM)403020100-50mV-55mVHolding Potential (mV)-65mV•This figure demonstrates the voltage-dependence of compound xpharmacology.•Compound x pharmacology is used as a measure of voltage control ineach day’s run

Sample ion channel recordingPre-compoundPost-compoundIC 50 (#1)Use-Dependency Ratio:IC 50 (#1) / IC 50 (#25)IC 50 (#25)

IonWorks can resolve differential pharmacology1 st Pulse Compound Concentration Response25 th Pulse Compound Concentration Response10010080Compound 1Compound 280Compound 1Compound 26060% Inhibition40200IC 50= 0.569µMIC 50= 121µM% Inhibition40200IC 50= 0.148µMIC 50= 50µM-20-20-400.01 0.1 1 10 100 1000Concentration (µM)0.01 0.1 1 10 100 1000 10000Concentration (µM)Two compounds with differential pharmacology, but similar use-dependency ratiosCompound 1 use-dependence ratio is 3.8Compound 2 use-dependence ratio is 2.5

Assay validation post-relocation• Run Patch Plate Tests– Verify overall operationalcapability• Run basic cell plates– Verify seal rates, sealvalues and current are onpar• Run protocol withreference agents– Verify IC 50 values haven’tdrifted

What happened?• Run protocol withreference agents– Verify IC 50 valueshaven’t drifted-- Compound IC 50 values shifted from historical-- cell-cell variability unacceptably high-- run-down/run-up outside of allowable limits (± 5%)

Resolution approachRe-chlorideE-headThaw new cellsSteps takento mitigateissuesLiaise with MDCObtainfreshreagentsPreparefreshsolutions

Further investigations…• Current-Voltage relationship•V 1/2 Inactivation• Quantify current drift (run-up/run-down)when comparing pre & post

I-V and inactivation curvesre-visited…1.2Normalized Current-Voltage Relationship0.2Normalized Current-Voltage RelationshipVoltage (mV)1.21.21.01.0Na v InactivationNa v InactivationNormalized Current Value1.00.80.60.40.20.0-70 -60 -50 -40 -30 -20 -10 10 20-0.2Peak = -10mVn=7Normalized Current Value-70 -60 -50 -40 -30 -20 -10 0 10 20Voltage (mV)-0.4-0.6-0.8-1.0-1.2Inactivation0.80.80.60.60.40.40.20.20.00.0-0.2-0.2V 1/2 Inactivation = -55mV ± 0.36V 1/2 Inactivation = -55mV ± 0.36n=7-100 -80 -60 -40 -20 0-100 -80 -60 -40 -20 0Voltage (mV)Voltage (mV)The current-voltage and steady-state inactivation relationships are shown here. Peak in the I-Vcurve is -10mV and the v 1/2 of inactivation is -55mV.

Voltage control?• Look at repetitive scans with multiple additionsCurrent - Voltage RelationshipVoltage (mV)-70 -60 -50 -40 -30 -20 -10 10 20no addition1st buffer addition2nd buffer addition-0.5-1.0Current (nA)-1.5-2.0-2.5Peak run-downThe figure here represents the current-voltage relationships following multiple additions of buffersolution

Pre- to post-addition current amplitudechanges12005.12.06 Na vChip 1 -55mV to 005.12.06 Na vChip 2 -100mV to 011.27.07 Na vChip 1 -55mV to -10mV11.27.07 Na vChip 2 -100mV to -10mV7010060Cell Number8060402000-56-1011-1516-2021-2526-3031-35Pre - Post Current Change (%)36-4041-4546-5051-5556-6061-6566-7071-7576-8081-8586-9091-9596-100Cell Number504030201000-56-1011-1516-2021-2526-3031-35Pre - Post Current Change (%)36-4041-4546-5051-5556-6061-6566-7071-7576-8081-8586-9091-9596-100The histograms here depict the trend in current amplitude change (absolute change) that is seenwhen comparing current recordings prior to and following a single buffer addition. Recordings wereobtained from holding potentials of either -55mV or -100mV.

Where we go from here…• Is this a cell line and/or hardware issue?– Assess alternate Na v cell line–Na v cell line on alternate platform– 12 vs. 48 channel dispense head?• Does a PPC mode assay reduce variability?• Re-clone cell line in CHO background• Shorten pulse train length and frequency• Continue to liaise with MDC

Summary• Cell line husbandry and detailed characterization ofgrowth parameters are critical to maximizing success• IonWorks is an integral part of a coordinated ionchannel drug discovery paradigm– Rapid SAR analysis and compound attrition– Ability to support high-speed chemistry approaches– Additive and complementary to optical approaches• Functional pharmacological data can be obtainedrapidly to aid in optimal candidate drug identification– Provides a means to identify compounds targeted to diseaserelevantion channel states

Acknowledgements• Peter Haddock• Chris Benjamin• Mark Weber• Jim Offord• Walt Volberg• Joe Mather• Kevin Allard• Jim Costantin•Richard Kondo

Back-up slides

IonWorks application in drug discovery casestudy #2Kv assay format• HT & PPC modes• 50:50Trypsin/Versene mixto lift cells• Amphotericin B togain access• Pulse to -20mV from-80mV for 3 sec.

Kv70Single hole mode70Population patch mode606030μM retagabinecontrol5050Cell count40304030controlactivation10μM XE991inhibition20200.2nA0.2nA10102s2s000.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2Kv current (nA)Population patch mode assay increases datafidelity and throughput 4xIonWorks assay can identify activators and inhibitors

Correlations between FLIPR and IonWorks aidin advancing drug candidacyFLIPR vs. IonWorks Correlation100FLIPR Na vIC 50(μM)1010.1 1 10 100IonWorks Na v25 th Pulse IC 50(μM)The strong correlation between the two platforms contributes support to the SARthat drives screening and project advancement.