The ipro. ipsc-derived cells going high throughput: new strategies for ion channels drug discovery. Silvia Cainarca
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1 The ipro ipsc-derived cells going high throughput: new strategies for ion channels drug discovery Silvia Cainarca Ncardia Applications Workshop November 30 th, 2017
2 Rationale: Early drug discovery, run in High Throughput Screening format, largely employs heterologous recombinant systems over-expressing a target of interest in a disease-unrelated host cell line An ideal pharmacological profiling would rely on testing the drug candidate against human disease-relevant cells but this has intrinsic limitations Human induced pluripotent stem cells (hipscs) represent a valid tool since they could be: obtained from patients in vitro cultured and expanded for long periods genetically modified differentiated into virtually any cell type Korean J Intern Med 2017;32(1): Furthermore, merging of optogenetics and ipsc-based technologies provides informative and cost-effective drug screening tool to achieve fast control of specific cell events -2- Silvia Cainarca - Ncardia Workshop 2017
3 Project Goals: Pharmacological characterization of human ipsc-derived neurons for high throughput drug screening (HTS), employing instrumentations and technologies commonly and broadly used for running HTS on recombinant cell lines Development of ipsc-derived opto-neurons suitable for high-throughput screening (HTS) HTS-compatible platform based on the use of optogenetics for drug screening, using human ipsc-derived cardiomyocytes -3- Silvia Cainarca - Ncardia Workshop 2017
4 Human ipsc-derived Neurons for High Throughput Drug Screening (HTS) -4- Silvia Cainarca - Ncardia Workshop 2017
5 Human ipsc-derived Neurons for HTS Workflow ipsc -derived Neurons 1 Assay set up 2 HTS assay optimization: Cell culture optimization: -culture protocol -coating -cell density -seeding time -microtiter plate format Assay optimization: -readout -reference compounds -instruments -adaptation from a semi-automated to a fully automated procedure 3 HTS and Hit Confirmation: -screening of SM library -data analysis -hit selection -hit confirmation -activity determination -5- Silvia Cainarca - Ncardia Workshop 2017
6 Human ipsc-derived Neurons for HTS: CNS.4U TM Morphological Analysis DIV1 DIV7 384 MTP 20X DIV14 20X DIV20 20X 20X -6- Silvia Cainarca - Ncardia Workshop 2017
7 Human ipsc-derived Neurons for HTS Functional Response Evaluation Test the activity of different key players, with a pivotal role in the pathophysiology of neuronal activity (such as Glutamate Receptors and Voltage-gated Ca 2+ Channels) in ipsc-derived neurons at FLIPR TETRA Challenging points Physiological expression level of the target protein Signal amplitude, robustness and reproducibility -7- Silvia Cainarca - Ncardia Workshop 2017
8 F /F 0 F /F 0 F /F 0 CNS.4U TM Neurons: Glutamate Receptors Ca 2+ - and MP-Assay: Preliminary Results D o s e r e s p o n s e c u r v e G lu ta m a te c /w D IV 7 D o s e r e s p o n s e c u r v e G lu ta m a te c /w D IV 1 4 D o s e r e s p o n s e c u r v e G lu ta m a te c /w D IV C a 2 + -d y e 1.5 C a 2 + -d y e 1.0 C a 2 + -d y e 0.5 M P -d y e 1.0 M P -d y e M P -d y e G lu ta m a te [M ] G lu ta m a te [M ] G lu ta m a te [M ] EC 50 Ca : dye nm EC 6.432e : 2.0 µm EC50 MP-dye 2.015e-006 EC 50 Ca : dye nm EC 9.159e : 1.5 µm EC50 MP-dye 1.533e-006 EC 50 Ca : dye µm EC 2.637e : 1.5 µm EC50 MP-dye 5.676e Silvia Cainarca - Ncardia Workshop 2017 GP
9 F /F 0 F /F 0 CNS.4U TM Neurons: NMDA Receptors Ca 2+ -Assay Preliminary Results 1.5 (R S )-(T e tra z o l-5 -y l)g ly c in e N M D A 0.8 C E R E S T A T C E R E S T 1.0 G lu ta m a te EC 50 : 191 nm EC 50 : 6.4 µm EC 50 : 1.23 µm C o n c e n tr a tio n [M ] C o n c e n tr a tio n [M ] EC50 (RS)-(Tetrazol-5-yl)glycine 1.914e-007 NMDA 6.414e-006 Glutamate 1.235e-006 IC 50 : 482 nm IC e-007 In presence of 31.6 µm NMDA Neurology -9- Silvia Cainarca - Ncardia Workshop 2017
10 F /F 0 F /F 0 F /F 0 CNS.4U TM Neurons: Voltage-gated Ca 2+ Channels Ca 2+ - and MP-Assay: Preliminary Results K + Activation Curve K + Activation Curve K + Activation Curve A c tiv a tio n C u r v e c /w D IV 7 C a 2 + -d y e M P -d y e A c tiv a tio n C u r v e A c tiv a tio n C u r v e c /w c /w D IV 1 4 D IV C a d y e M P -d y e C a 2 + -d y e M P -d y e K + [M ] K + [M ] K + [M ] EC50 Ca 2+ -dye MP-dye EC 50 : 15.8 mm EC 50 : 34 mm EC50 Ca 2+ -dye MP-dye EC 50 : 18.6 mm EC 50 : 33 mm EC50 Ca 2+ -dye MP-dye EC 50 : 20 mm EC 50 : 50 mm The functional response of the channels was evaluated by applying increasing concentration of K + to activate the channels -10- Silvia Cainarca - Ncardia Workshop 2017 GP
11 F /F 0 K + Activation Curve CNS.4U TM Neurons: L-Type CaV Channels K + Activation and Inactivation Curve: Preliminary Results K + Inactivation Curve Promising preliminary results indicating the possibility to use these cells in HTS Could this process be improved? Nicardipine R e s tin g Half inactivated Optogenetics: The Use of Light to set up a High-Throughput Screening Assay 0.0 EC C o n c e n tr a tio n [M ] Resting IC 50 : 1.02 µm Half inactivated IC 50 : < 100 nm NICARDIPINE resting 1.023e-006 NICARDIPINE half inact 2.946e Silvia Cainarca - Ncardia Workshop 2017
12 Optogenetics basic concepts Sensors translate cell physiological signals into optical signals Actuators transduce optical signals into physiological signals Less toxic than organic dye The possibility to have optogenetics sensors able to detect intracellular Ca 2+ influxes and actuators such as a channelrhodopsin for a fine tuned light-mediated electrical modulation of cell events could allow to control and record Ca 2+ variation in a spatiotemporalspecific manner Fast and reversible stimulation -12- Silvia Cainarca - Ncardia Workshop 2017
13 Genetically Encoded Optogenetics Proteins: the Evolution Chrimson GCaMP6f Dufour et al. Neurophotonics,
14 Development of ipsc-derived Opto-Neurons Workflow for CRISPR/Cas9 Genome Editing - In silico design of sgrna and HDR donor - Sequence analysis of genomic target region Construct Generation: - CRISPR sgrnas/cas9 - HDR donor Transfection of hipscs Recovery, enrichment and plating at low density Selection of clones Screening of clones and characterization 14 KM, CK
15 EuroTransBio: 11 th Transnational Call Grant Title Partner Axxam contribution in ipsc NeurOptics Development of ipscs derived Optoneurons suitable for high-throughput screening (HTS) Ncardia Generation of Opto-iPSC Validation of Opto- Neurons for HTS GCaMP6f -15- Silvia Cainarca - Ncardia Workshop 2017
16 HTS-compatible Platform-based on the Use of Optogenetics for Drug Screening: ipsc-derived Cardiomyocytes -16- Silvia Cainarca - Ncardia Workshop 2017
17 Voltage-gated Ion Channel Light-pacing by OPTO-FLIPR % ΔF (membr. potential DYE) Standard FLIPR TETRA + Custom-made modification = «OPTO FLIPR» 2 exc/em Alternated sampling 2 exc/em Flexible temporal pattern Robust and specific Na V 1.x pacing by fast kinetics ChR2 Stimulus A Syncytium assays Target A CX-43 Target B Ions / small molecules No blocker Cell line A Cell line B ChR2 exc Dye imaging Amitriptyline 30µM Functional response of «Target A» (which includes changes in ion or small molecule concentrations) can be transferred to «Cell line B» through Gap Junctions (Connexin proteins) and can modulate the activity of «Target B», or can be monitored by Genetically encoded indicators expressed in «Cell line B» unpublished VA -17- Silvia Cainarca - Ncardia Workshop 2017
18 Voltage-gated Ion Channel Light-pacing by OPTO-FLIPR % ΔF (membr. potential DYE) Standard FLIPR TETRA + Custom-made modification = «OPTO FLIPR» 2 exc/em Alternated sampling 2 exc/em Flexible temporal pattern Robust and specific Na V 1.x pacing by fast kinetics ChR2 Use-dependent Syncytium blockers assays validated with Ca V 1.x pacing by fast kinetics ChR2 (syncytium assay) Stimulus A Target A CX-43 Target B Ions / small molecules No blocker Cell line A Not use-dependent blocker Cell line B ChR2 exc Dye imaging Amitriptyline 30µM Functional response of «Target A» (which includes changes in ion or small molecule concentrations) can be transferred to «Cell line B» through Gap Junctions (Connexin proteins) and can modulate the activity of Use-dependent blocker «Target B», or can be monitored by Genetically encoded indicators expressed in «Cell line B» unpublished VA -18- Silvia Cainarca - Ncardia Workshop 2017
19 % ΔF (calcium DYE) % ΔF (calcium DYE) % ΔF (calcium DYE) OPTO-cardiac Safety with Light-paced ipsc-derived Cardiomyocytes Standard FLIPR «OPTO-FLIPR» Cor.4U Cor.4U + HEK/ChR2 fast (syncytium assay) GOOD spontaneous activity Perfect synchronization & Light-pacing up to 1.5 Hz Control (28 bpm; +40% ΔF) 0.5 Hz Frequency / use-dependent blocker Isoproterenol 1µM (44 bpm; +50% ΔF) 1 Hz (1 well replicate) No synchronization 1.5 Hz No blocker -19- (4 wells replicate) Silvia Cainarca - Ncardia Workshop 2017 unpublished (4 wells replicate) VA
20 EuroTransBio: 10 th Transnational Call Grant Title Partners OPTEL A novel optogenetic electrophysiology platform for ion channel and transporter screening Nanion Technologies GmbH University of Bonn Axxam contribution in ipsc Optogenetic pacing of Cor.4U cardiomyocytes: gene transfer of ChR2 using adeno-associated viruses or co-culture with ChR2-expressing HEK293 cells End: Nov Silvia Cainarca - Ncardia Workshop 2017 VA
21 Concluding Remarks Opto-Neurons in HTS The functional presence of glutamate receptors was shown using both a Ca 2+ and membrane potential dye Among the different glutamate receptor it was demonstrated the presence of NMDAR, using specific agonists and antagonist Applying different concentration of K + it was possible to measure the presence of voltagegated Ca 2+ channels and in particular of the L-type Ca 2+ channels by testing specific state dependent blocker It is ongoing the generation of an optoneurons system, for a fast and less toxic Ca 2+ measurement and for setting up fine tuned light-mediated use/state-dependency FLIPR TETRA assays Light-paced ipsc-derived Cardiomyocytes Syncytium assays validated for optogenetic control of recombinant / native ion channel function ChR2 light-pacing of: Recombinant voltage-gated Na + or Ca 2+ channels ipsc-derived cardiomyocytes beating up to 1.5 Hz Frequency / use-dependency studies enabled -21- Silvia Cainarca - Ncardia Workshop 2017
22 and all of you for your attention! Aknowledgments Lucia Rutigliano (ipsc lab - TS group) Giuliana Piazza (ST group) Viviana Agus (CB group) Katharina Montag (MB - TS group) Christina Kuhn (MB - TS group) Michela Stucchi (PI NeurOptics - ST group head) Riccardo Rizzetto (PI Optel - Ephys group) Corporate Offices & Laboratories Via Meucci , Bresso/Milan (Italy) Cambridge Innovation Center 1 Broadway, Cambridge, Mass (USA) Contacts Dr Silvia Cainarca silvia.cainarca.sc@axxam.com Dr. Sabrina Corazza sabrina.corazza.sc@axxam.com Dr. Lia Scarabottolo lia.scarabottolo.ls@axxam.com Claudia Caserini (TS group head) Lia Scarabottolo (Director Discovery Services) Stefan Lohmer (CEO)
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