The Application of Cell-Based Impedance Technology in Drug Discovery Yama A. Abassi, PhD Sr. Director Cell Biology and Assay Development ACEA Biosciences www.aceabio.com
ACEA Biosciences � Founded in early 2002 � 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 2004 � In November of 2007 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 www.aceabio.com
xCELLigence RTCA SP System E-Plate Computer and Software Gold Microelectrode Analyzer Covers 80% of Well Area Plate Reader(RTCA SP) (in CO2 incubator) www.aceabio.com
Electronic Sensor Technology Applied to Electronic Sensor Technology Applied to Cell Biology: Principle of Operation Cell Biology: Principle of Operation Derivati Derivation of Cell Index n 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: 1. When cells are not present or are not well-adhered on the electrodes, then the CI is zero 2. 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. 3. Additionally, change in a cell status, such as cell morphology, cell adhesion or cell viability will lead to a change in CI. www.aceabio.com
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 www.aceabio.com
Cell-based Assays: Traditional Methods Traditional methods: Seed Treatment: Labeling (e.g. optical) cells e.g.: drug compound & End-point Measurement Data Black Box 18-24 Hours 24-48 Hours 1-24 Hours Initiate Analysis Experiment www.aceabio.com
Cell-based Assays: Traditional Methods vs xCELLigence System Traditional methods: Seed Treatment: Labeling (e.g. optical) cells e.g.: drug compound & End-point Measurement Data 18-24 Hours 24-48 Hours 1-24 Hours Initiate Analysis Experiment 7 Control 6 Cmpd A x e Cmpd B d 5 ll In Cmpd C e 4 C Cmpd D d lize 3 Treatment a rm 2 o N 1 0 0 10 20 30 40 50 60 70 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 www.aceabio.com captured
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 www.aceabio.com
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 www.aceabio.com
Time-Dependent Cell Response Profiling www.aceabio.com
The Road to Cellular Cytotoxicity Takes Many Twists and Turns Proteasome Inhibitor N-Glycosylation Inhibitor 5 DMSO 2.5 Tunicamycin 0.125 uM 0.25 uM 4 Normalized Cell Index 0.5 uM 2 1 uM 10 uM" N orm a lize d C I 2 uM 4 uM 3.3 uM 3 1.5 8 uM 1.1 uM 1 0.33 uM 2 Control 0.5 1 0 0 0 10 20 30 40 50 60 0 10 20 30 40 50 60 70 80 Time (hours) Time (hours) Anti-Mitotic DNA Damaging 3.5 10 uM 10 uM 3 1 7 1.1 uM Norm alized CI 2.5 0.12 6 0.12 uM Normalized CI 5 2 Ctrl 0.0137 uM 4 0.0016 uM 1.5 Ctrl 3 1 2 0.5 1 0 0 0 20 40 60 80 100 0 20 40 60 80 100 120 Time (hours) Time (hours) www.aceabio.com
Are Impedance-Based Cell Response Profiles Predictive of Biological Mechanism ? www.aceabio.com
TCRP Approach O O E-Plate O OH HO OH Add Add Monitor Monitor Compound Cells 48 hours 20-24 hours Interdigitated gold microelectrodes Real-Time Continuous Monitoring Impedance-based real-time cellular response profile Seed 4000 A549 Cancer Cells in 96 well E-Plates Treat with Compounds at a final concentration Of 20 μ M Spectrum Compound Library from MS Discovery (Collection of FDA approved drugs, nature compounds Experimental compounds, insecticides and herbicides) Monitor the cellular response for 48 hours www.aceabio.com Compare cytological profiles
Hit Selection and Clustering Analysis TCRP from Screen Short-term response Long Term Response (w/in 1 hour) (1-48 hours) Hit criteria Hit criteria (25% of control) (25% and 40%) www.aceabio.com
TCRP with Known Mechanisms Short-Term Response Long-Term Response Anti-mitotics Protein-Synthesis Anti-Histamine 3.5 4 Ctrl 4 Ctrl Ctrl 3 Normalized CI Colchicine Normalized CI Normalized CI 3 Emetine 3 2.5 Azelastine 2 2 2 1.5 1 1 1 0.5 0 0 0 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 Time (hours) Time (hours) Time (hours) Nuclear Hormone 7 DNA Damaging Serotonin Receptor Antagonist 6 Ctrl 5 Normalized CI 5 5 Hydrocortisone Normalized CI Ctrl Ctrl 4 Normalized CI 4 4 Etoposide 3 Methiothepin 3 3 2 2 2 1 1 1 0 0 0 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 Time (hours) Time (hours) Time (hours) L-Type Voltage-gated Ca HDAC Inhibitors Channel Inhibitor Ctrl 7 4 Trichostatin A Ctrl 6 Normalized CI Normalized CI 5 Amlodipine 3 4 2 3 2 1 1 0 0 0 20 40 60 80 100 0 20 40 60 80 Time (hours) Time (hours) www.aceabio.com
TCRP with Known Mechanisms Short-Term Response Long-Term Response Anti-mitotics Protein-Synthesis Anti-Histamine 3.5 4 Ctrl 4 Ctrl Ctrl 3 Normalized CI Normalized CI Colchicine Normalized CI 3 Emetine 3 2.5 Azelastine 2 2 2 1.5 1 1 1 0.5 0 0 0 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 Time (hours) Time (hours) Time (hours) Nuclear Hormone 7 DNA Damaging Serotonin Receptor Antagonist 6 Ctrl 5 Normalized CI 5 5 Hydrocortisone Normalized CI Ctrl Ctrl 4 Normalized CI 4 4 Etoposide 3 Methiothepin 3 3 2 2 2 1 1 1 0 0 0 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 Time (hours) Time (hours) Time (hours) L-Type Voltage-gated Ca HDAC Inhibitors Channel Inhibitor Ctrl 7 4 Trichostatin A Ctrl 6 Normalized CI Normalized CI 5 Amlodipine 3 4 2 3 2 1 1 0 0 0 20 40 60 80 100 0 20 40 60 80 Time (hours) Time (hours) www.aceabio.com
TCRP of Anti-mitotic Compounds www.aceabio.com
Characterization of Impedance- Based Anti-mitotic Profile Ctrl 14 h (Phase II) 6 h (Phase I) 48 h (Phase IV) 24 h (Phase III) Mitotic Index 90 80 Untreated 70 Mitotic Index (%) 12.5 nM Paclitaxel 60 50 40 30 20 10 0 0 20 40 60 80 Time (h) www.aceabio.com
Validation of Mitotic Arrest Profile Eg5 Small Molecule Inhibitor 7 S-Trityl-L-Cysteine 6 DMSO 5 Normalized CI 4 3 2 1 0 0 20 40 60 80 100 Time (h) S-Trityl-L-Cysteine Ctrl anti- tubulin Ab anti- PH3 www.aceabio.com
Compounds with Anti-mitotic Profile from the Spectrum Collection www.aceabio.com
Systematic Analysis of Impedance-Based Cell Response Profiles www.aceabio.com
Curve Classification Algorithm and Display www.aceabio.com
Protein Synthesis In Nuclear Receptor DNA Damaging Clustering Analysis Ca Modulator Anti-mitotic CELECOXIB TAMOXIFEN FLUPHENAZINE PERPHENAZINE METHIOTHEPIN COLCHICINE NOCODAZOLE NOSCAPINE beta-PELTATIN ESTRADIOL PUROMYCIN PYRROMYCIN CYCLOHEXIMIDE EMETINE LYCORINE TENIPOSIDE CAMPTOTHECIN STROPHANTHIDIN OLEANDRIN ETOPOSIDE BUDESONIDE HOMATROPINE HYDRALAZINE HYDROCORTISONE METHYLPREDNISOLONE www.aceabio.com
TCRP COX-2 Inhibitors Rofecoxib Valdecoxib COX-2 Inhibitors O 5 20 uM 20 uM 5 N O O 10 uM 4 10 uM 4 Normalized CI Normalized CI 5 uM 5 uM 3 3 2.5 uM Ctrl 2 2 1 1 O S O S O 0 0 O H 2 N Valdecoxib 0 20 40 60 80 Rofecoxib 0 20 40 60 80 Time (hours) Time (hours) Deracoxib F F F F 5 F 20 uM 4 10 uM Normalized CI N N F N 5 uM N 3 2.5 uM 2 O 1 O S S 0 O O O NH 2 NH 2 0 20 40 60 80 Deracoxib Celecoxib Time (hours) www.aceabio.com
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