The Application of Cell-Based Impedance Technology in Drug Discovery

Transcription

1 The Application of Cell-Based Impedance Technology in Drug Discovery Yama A. Abassi, PhD Sr. Director Cell Biology and Assay Development ACEA Biosciences

2 ACEA Biosciences Founded in early Located in San Diego, CA Mission: Integration of microelectronics with cell biology and molecular biology for providing innovative and cost-effective microelectronic biological analysis systems and applications for life science industry and clinical diagnostics ACEA s first product was marketed under the brand name of RT-CES system in In November of 7 Roche and ACEA Biosciences entered into an exclusive agreement for the development, supply and distribution for ACEA Bioscience s real-time cell assay technology. Under the terms of the agreement, RAS will exclusively market systems for real-time cell analysis, based on ACEA Bioscience's impedance-based technology The first joint Roche/ACEA product is marketed under the brand name of xcelligence

3 xcelligence RTCA SP System Computer and Software E-Plate Analyzer Plate Reader(RTCA SP) (in C incubator) Gold Microelectrode Covers 8% of Well Area

4 Electronic Sensor Technology Applied to Cell Biology: Principle of peration Derivation of Cell Index A dimensionless parameter termed Cell Index (CI) is derived as a relative change in measured electrical impedance to represent cell status. Several features of the CI are summarized:. When cells are not present or are not well-adhered on the electrodes, then the CI is zero. Under the same physiological conditions when more cells are attached on the electrodes, then the CI values are larger. Thus, CI is a quantitative measure of cell number present in a well.. Additionally, change in a cell status, such as cell morphology, cell adhesion or cell viability will lead to a change in CI.

5 Advantages of xcelligence System for Cell-Based Assays and Drug Discovery Applications Label free, no reporters Non-invasive measurement Real-time Monitoring -Short-term (milliseconds) -Long-term (days and weeks) Continuous QC

6 Cell-based Assays: Traditional Methods Seed cells Treatment: e.g.: drug compound Traditional methods: Labeling (e.g. optical) & End-point Measurement Initiate Experiment 8- Hours Black Box -8 Hours - Hours Data Analysis

7 Cell-based Assays: Traditional Methods vs xcelligence System Seed cells Treatment: e.g.: drug compound Traditional methods: Labeling (e.g. optical) & End-point Measurement Initiate Experiment Normalized Cell Index Hours -8 Hours - Hours Control Cmpd A Cmpd B Cmpd C Cmpd D Treatment Data Analysis Time (Hours) Continuous QC xcelligence System : Label-free: Electronics-based detection Real-time: Continuous measurement, data analysis and display Therefore, both short term and long term compound effects can be captured

8 Applications Developed on the xcelligence System Cell Proliferation Cell Quality Compound-mediated Cytotoxicity Cell-mediated Cytotoxicity Cell Adhesion and Spreading Functional Monitoring of Receptor Tyrosine Kinase Signaling Functional Monitoring of GPCR Signaling IgE Receptor Function Cell Invasion and Migration Barrier Function Viral Cytopathogenecity

9 Applications Developed on the xcelligence System Cell Proliferation Cell Quality Compound-mediated Cytotoxicity Cell Response Profiling Cell-mediated Cytotoxicity Cell Adhesion and Spreading Functional Monitoring of Receptor Tyrosine Kinase Signaling Functional Monitoring of GPCR Signaling IgE Receptor Function Cell Invasion and Migration Barrier Function Viral Cytopathogenecity

10 Time-Dependent Cell Response Profiling

11 The Road to Cellular Cytotoxicity Takes Many Twists and Turns Normalized Cell Index DMS.5 um.5 um.5 um um um um 8 um um. um. um.7 um.6 um Proteasome Inhibitor Anti-Mitotic N-Glycosylation Inhibitor um". um. um. um Control Tunicamycin um DNA Damaging 6 8

12 Are Impedance-Based Cell Response Profiles Predictive of Biological Mechanism?

13 H H H TCRP Approach E-Plate Add Cells Monitor - hours Add Compound Monitor 8 hours Interdigitated gold microelectrodes Real-Time Continuous Monitoring Impedance-based real-time cellular response profile Seed A59 Cancer Cells in 96 well E-Plates Treat with Compounds at a final concentration f μm Spectrum Compound Library from MS Discovery (Collection of FDA approved drugs, nature compounds Experimental compounds, insecticides and herbicides) Monitor the cellular response for 8 hours Compare cytological profiles

14 Hit Selection and Clustering Analysis TCRP from Screen Short-term response (w/in hour) Long Term Response (-8 hours) Hit criteria (5% of control) Hit criteria (5% and %)

15 TCRP with Known Mechanisms Short-Term Response Anti-Histamine Azelastine 6 8 Serotonin Receptor Antagonist 5 Methiothepin 6 8 L-Type Voltage-gated Ca Channel Inhibitor Amlodipine Colchicine 6 8 Etoposide Anti-mitotics DNA Damaging Long-Term Response HDAC Inhibitors Trichostatin A Emetine 6 8 Hydrocortisone 6 8 Protein-Synthesis Nuclear Hormone 6 8

16 TCRP with Known Mechanisms Short-Term Response Anti-Histamine Azelastine 6 8 Serotonin Receptor Antagonist 5 Methiothepin 6 8 L-Type Voltage-gated Ca Channel Inhibitor Amlodipine Colchicine 6 8 Etoposide Anti-mitotics DNA Damaging Long-Term Response HDAC Inhibitors Trichostatin A Emetine 6 8 Hydrocortisone 6 8 Protein-Synthesis Nuclear Hormone 6 8

17 TCRP of Anti-mitotic Compounds

18 Characterization of Impedance- Based Anti-mitotic Profile 6 h (Phase I) h (Phase II) h (Phase III) 8 h (Phase IV) Mitotic Index Mitotic Index (%) 9 8 Untreated nm Paclitaxel Time (h)

19 Validation of Mitotic Arrest Profile Eg5 Small Molecule Inhibitor 7 6 S-Trityl-L-Cysteine 5 DMS 6 8 Time (h) S-Trityl-L-Cysteine antitubulin Ab anti- PH

20 Compounds with Anti-mitotic Profile from the Spectrum Collection

21 Systematic Analysis of Impedance-Based Cell Response Profiles

22 Curve Classification Algorithm and Display

23 Ca Modulator Anti-mitotic Protein Synthesis In DNA Damaging Nuclear Receptor Clustering Analysis CELECXIB TAMXIFEN FLUPHENAZINE PERPHENAZINE METHITHEPIN CLCHICINE NCDAZLE NSCAPINE beta-peltatin ESTRADIL PURMYCIN PYRRMYCIN CYCLHEXIMIDE EMETINE LYCRINE TENIPSIDE CAMPTTHECIN STRPHANTHIDIN LEANDRIN ETPSIDE BUDESNIDE HMATRPINE HYDRALAZINE HYDRCRTISNE METHYLPREDNISLNE

24 TCRP CX- Inhibitors CX- Inhibitors N S H N Valdecoxib S F F F F F N N F N N S S NH NH Rofecoxib Celecoxib Deracoxib Valdecoxib 5 um um 5 um 6 8 Rofecoxib 5 um um 5 um.5 um 6 8 Deracoxib 5 um um 5 um.5 um 6 8

25 TCRP CX- Inhibitors CX- Inhibitors N S H N Valdecoxib S Rofecoxib Valdecoxib 5 um um 5 um 6 8 Rofecoxib 5 um um 5 um.5 um 6 8 F N N S NH Celecoxib F F F F F N N S NH Deracoxib Celecoxib 5 um um 5 um.5 um 6 8 Deracoxib 5 um um 5 um.5 um 6 8

26 Validation of Celecoxib as Modulator of Intracellular Calcium Levels. BAPTA AM 5 um Celecoxib um BAPTA AM DMS Untreated

27 Validation of Celecoxib as Modulator of Intracellular Calcium Levels BAPTA AM 5 um Celecoxib um BAPTA AM DMS Untreated Mean Fluorescence (Arbitrary Units) 5 - Thapsigargin 5 um 5 um.5 um 6.5 um Celecoxib

28 Advantages of Short Term and Long Term Monitoring of Cellular Response Profiles H HeLa Cells 5 S HN N H CH Monastrol CH Monastrol 5 6

29 Advantages of Short Term and Long Term Monitoring of Cellular Response Profiles H HeLa Cells 5 S HN N H CH Monastrol CH Monastrol 5 6

30 Advantages of Short Term and Long Term Monitoring of Cellular Response Profiles H HeLa Cells 5 S HN N H CH Monastrol CH Monastrol 5 6

31 Advantages of Short Term and Long Term Monitoring of Cellular Response Profiles H HeLa Cells 5 S HN N H CH Monastrol CH Monastrol 5 6 Long Term Monastrol Phospho-Histone H

32 Advantages of Short Term and Long Term Monitoring of Cellular Response Profiles H HeLa Cells N 5 S HN N H CH Monastrol CH Monastrol 5 6 H CC HC N H Long Term CH nifedipine Monastrol Phospho-Histone H

33 Advantages of Short Term and Long Term Monitoring of Cellular Response Profiles S HN N H H CH Monastrol Mean Relative Fluorescence 6 8 CH Short Term 5 Monastrol Voltage-Gated Calcium Level Modulation 6 8 Monastrol (um) HeLa Cells 5 6 H CC HC Long Term Phospho-Histone H N H Monastrol N CH nifedipine

34 Functional Monitoring of GPCR Signaling

35 GPCR Activation Leads to Modulation of the Actin Cytoskeleton

36 Dynamic Monitoring of GPCR-mediated Morphological Dynamics Using the xcelligence RTCA System Histamine Phalloidin anti-paxillin.8 CTR.6.. Unstimulated Time (h) +Histamine.5 Vasopressin Phalloidin anti-paxillin...9 CTR Unstimulated Vasopressin Time (h)

37 Functional Monitoring of Gq Coupled Receptors on the xcelligence RT-CA System Histamine H Receptor RT-CA Assay H-IP Assay His um His nm His pm CTR EC 5 =.7 nm [ H]IP bound (cpm) 5 5 EC 5 = 9 nm Time (hr) [Histamine] Log M [Histamine] Log M

38 Functional Monitoring of G s Coupled Receptors on the xcelligence RT-CA System Dopamine Receptor RT-CA Assay camp Assay...8 SKF 89, um SKF 89, nm SKF 89,. nm CTR 6 8 Time (hr) normalized CI EC 5 =.7 nm [SKF 89] Log M [camp] pmoles/well EC 5 =.9 nm [SKF 89] Log M

39 Functional Monitoring of G i Coupled Receptors on the xcelligence RT-CA System 5-HTA Receptor RT-CA Assay camp Assay H-DPAT, um 8-H-DPAT, nm 8-H-DPAT,. nm CTR 6 8 Time (hr).... EC 5 = 9 nm [8-H DPAT] Log M [camp], nmol/well 5 5 EC 5 = 6 nm [8-H-DPAT] Log M

40 Dynamic Monitoring of Endogenous Receptors Using the xcelligence System Histamine Receptor (Gq) in HeLa Cells Histamine um Histamine 6 nm Histamine nm CTR 6 8 Time (hr) Calcitonin Receptor (Gs) in CH Cells 6 8 Time (hr) Calcitonin 5 um Calcitonin nm Calcitonin pm CTR pioid Receptor (Gi) in NIE5 Cells DPDPE um DPDPE nm DPDPE pm CTR Time (hr) [Histamine] Log M [Calcitonin] Log M EC 5 = 6nM EC 5 = 85 pm EC 5 = nm [DPDPE] Log M

41 Dynamic Monitoring of Receptors in Disease-Relevant Cell Types Cor.AT Cells from Axiogenesis Mouse ES Cell-Derived Cardiomyocytes % Pure Population β Adrenergic Receptor Activation in Cardiomyocyte-Differentiated Cor.AT Cells Time (Hours) 75 nm 5 nm nm 6 nm. nm. nm EC-5=. nm

42 Identification of Histamine H Receptor Inverse Agonist using the xcelligence RT-CA System Normarlized CI Histamine nm CTR Time (h) Norm alized CI Histamine nm Loratidine um Time (h)

43 List of Receptors Functionally Monitored by the xcelligence GPCR RTK FcR (IgE and IgG) TCR System Death Receptors (FasR, TNFR) Integrins Toll Receptors Nuclear Hormone Receptors

44 Summary Impedance-based monitoring of cellular status using the xcelligence platform allows for monitoring of both short term and long term responses The ability to monitor short and long term responses within the same experiment provides cytological profiles which can be predictive of mechanism of action The non-invasive nature of impedance readout provides the advantage of working with primary cells or disease relevant cells both for long term cytotoxicity studies and short term receptor responses