Label‐Free Dynamic Mass Redistribution and Bio‐Impedance Methods for Drug Discovery

Manuel Grundmann1

1 Section Cellular, Molecular and Pharmacobiology, Institute for Pharmaceutical Biology, University of Bonn, Bonn
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 9.24
DOI:  10.1002/cpph.24
Online Posting Date:  June, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Label‐free biosensors are increasingly employed in drug discovery. Cell‐based biosensors provide valuable insights into the biological consequences of exposing cells and tissues to chemical agents and the underlying molecular mechanisms associated with these effects. Optical biosensors based on the detection of dynamic mass redistribution (DMR) and impedance biosensors using cellular dielectric spectroscopy (CDS) capture changes of the cytoskeleton of living cells in real time. Because signal transduction correlates with changes in cell morphology, DMR and CDS biosensors are exquisitely suited for recording integrated cell responses in an unbiased, yet pathway‐specific manner without the use of labels that may interfere with cell function. Described in this unit are several experimental approaches utilizing optical label‐free system capturing dynamic mass redistribution (DMR) in living cells (Epic System) and an impedance‐based CDS technology (CellKey). In addition, potential pitfalls associated with these assays and alternative approaches for overcoming such technical challenges are discussed. © 2017 by John Wiley & Sons, Inc.

Keywords: label‐free; biosensor; DMR; CDS; phenotypic assay

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Standard Dynamic Mass Redistribution Assay (DMR) Assay
  • Alternate Protocol 1: DMR Assay With Cell Suspensions
  • Alternate Protocol 2: Antagonist‐Mode DMR Assay
  • Basic Protocol 2: Standard Bio‐Impedance (CDS) Assay Protocol
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Standard Dynamic Mass Redistribution Assay (DMR) Assay

  Materials
  • HEK293 cells expressing the FFA2 receptor (FFA2‐HEK; contact Evi Kostenis at kostenis@uni‐bonn.de for cell line requests) growing in T‐75 (75‐cm2) flasks
  • Complete growth medium (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.05% (w/v) trypsin/0.02% (w/v) EDTA (e.g., Gibco, Thermo Fisher Scientific)
  • Assay buffer (see recipe)
  • 1 M propionic acid stock solution, aqueous (e.g., Sigma‐Aldrich)
  • Controls (see annotation to step 14, below)
  • 384‐well, fibronectin‐coated biosensor plate (e.g., Corning Cell Assay Plate 5042)
  • Multichannel and/or repeat pipettors
  • Multichannel manifold attached to cell culture vacuum pump (e.g., from Socorex, see Fig.  ; alternatively, use a liquid handling station that can be used for washing protocols, e.g., PerkinElmer JANUS automated workstation)
  • Centrifuge
  • DMR reader (e.g., Corning Epic BT reader or PerkinElmer EnSight)
  • 384‐well storage plates (e.g., Corning Costar 3657)
  • Liquid‐handling station capable of transferring all 384‐well formats at the same time (e.g., CyBi‐SELMA, Analytik Jena)
  • Incubator for label‐free readers without an on‐board temperature control unit (e.g., from Memmert)
  • PC workstation for processing label‐free data running e.g., EpicImager (Corning) or Kaleido Data Acquisition and Analysis Software (PerkinElmer), Microsoft Excel, and GraphPad Prism
  • Additional reagents and equipment for cell culture including trypsinization and cell counting (Phelan & May, 2016)

Alternate Protocol 1: DMR Assay With Cell Suspensions

  Additional Materials (also see protocol 1)
  • At least two ligands (antagonist, e.g. CATPB or GLPG0974, and an established agonist, e.g., propionic acid, acetic acid, 4‐CMTB)
  • Two 384‐well storage plates (e.g., Corning Costar 3657)

Alternate Protocol 2: Antagonist‐Mode DMR Assay

  Materials
  • 0.1 mg/ml poly‐D‐lysine (PDL)
  • Phosphate‐buffered saline (PBS; see recipe)
  • HEK293 cells expressing the FFA2 receptor (FFA2‐HEK; see Grundmann 2016) growing in T‐75 (75‐cm2) flasks
  • 0.05% (w/v) trypsin/0.02% (w/v) EDTA (e.g., Gibco, Thermo Fisher Scientific)
  • Complete growth medium (see recipe)
  • Assay buffer (see recipe)
  • 1 M propionic acid stock solution, aqueous (e.g., Sigma‐Aldrich)
  • Controls (see annotation to step 15, below)
  • 384‐well cellular dielectric spectroscopy biosensor plate (e.g., CellKey Assay Plate, Molecular Devices)
  • Multichannel and/or repeat pipettors
  • Multichannel manifold attached to cell culture vacuum pump (e.g., from Socorex, see Fig.  ; alternatively, use a liquid handling station that can be used for washing protocols, e.g., PerkinElmer JANUS automated workstation)
  • Centrifuge
  • 384‐well storage plate (e.g., Corning Costar 3657)
  • Bio‐impedance CDS reader (e.g., Molecular Devices CellKey System)
  • PC workstation to process label‐free data (e.g., Molecular Devices CellKey 384 Software, Microsoft Excel, GraphPad Prism)
  • Additional reagents and equipment for cell culture including trypsinization and cell counting (Phelan & May, 2016)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Aplin, A., Howe, A., Alahari, S., & Juliano, R. (1998). Signal transduction and signal modulation by cell adhesion receptors: The role of integrins, cadherins, immunoglobulin‐cell adhesion molecules, and selectins. Pharmacological Reviews, 50(2), 197–264.
  Arthofer, E., Hot, B., Petersen, J., Strakova, K., Jager, S., Grundmann, M., … Schulte, G. (2016). WNT stimulation dissociates a Frizzled 4 inactive state complex with Gα12/13. Molecular Pharmacology, 90(4), 447–459. doi: 10.1124/mol.116.104919.
  Blättermann, S., Peters, L., Ottersbach, P. A., Bock, A., Konya, V., Weaver, C. D., … Luschnig, P. (2012). A biased ligand for OXE‐R uncouples Gα and Gβγ signaling within a heterotrimer. Nature Chemical Biology, 8(7), 631–638. doi: 10.1038/nchembio.962.
  Bosco, M. D., Mohanasundaram, D. M., Drogemuller, C. J., Lang, C. J., Zalewski, P. D., & Coates, P. T. (2010). Zinc and zinc transporter regulation in pancreatic islets and the potential role of zinc in islet transplantation. The Review of Diabetic Studies, 7(4), 263. doi: 10.1900/RDS.2010.7.263.
  Chen, K., Obinata, H., & Izumi, T. (2010). Detection of G protein‐coupled receptor‐mediated cellular response involved in cytoskeletal rearrangement using surface plasmon resonance. Biosensors and Bioelectronics, 25(7), 1675–1680. doi: 10.1016/j.bios.2009.12.006.
  Concepcion, J., Witte, K., Wartchow, C., Choo, S., Yao, D., Persson, H., … Ma, W. (2009). Label‐free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization. Combinatorial Chemistry & High Throughput Screening, 12(8), 791–800. doi: 10.2174/138620709789104915.
  Du, Y., Li, Z., Li, L., Chen, Z., Sun, S.‐Y., Chen, P., … Fu, H. (2009). Distinct growth factor‐induced dynamic mass redistribution (DMR) profiles for monitoring oncogenic signaling pathways in various cancer cells. Journal of Receptors and Signal Transduction, 29(3–4), 182–194. doi: 10.1080/10799890902976933.
  Eder, J., Sedrani, R., & Wiesmann, C. (2014). The discovery of first‐in‐class drugs: Origins and evolution. Nature Reviews Drug Discovery, 13(8), 577–587. doi: 10.1038/nrd4336.
  Fang, Y. (2014). Label‐free drug discovery. Frontiers in Pharmacology, 5, 52. doi: 10.3389/fphar.2014.00052.
  Fang, Y., Ferrie, A. M., Fontaine, N. H., Mauro, J., & Balakrishnan, J. (2006). Resonant waveguide grating biosensor for living cell sensing. Biophysical Journal, 91(5), 1925–1940. doi: 10.1529/biophysj.105.077818.
  Fang, Y., Ferrie, A. M., Fontaine, N. H., & Yuen, P. K. (2005). Characteristics of dynamic mass redistribution of epidermal growth factor receptor signaling in living cells measured with label‐free optical biosensors. Analytical Chemistry, 77(17), 5720–5725. doi: 10.1021/ac050887n.
  Fatehullah, A., Tan, S. H., & Barker, N. (2016). Organoids as an in vitro model of human development and disease. Nature Cell Biology, 18(3), 246–254. doi: 10.1038/ncb3312.
  Fellmann, C., Gowen, B. G., Lin, P.‐C., Doudna, J. A., & Corn, J. E. (2017). Cornerstones of CRISPR‐Cas in drug discovery and therapy. Nature Reviews Drug Discovery, 16, 89–100. doi: 10.1038/nrd.2016.238.
  Fogel, R., Limson, J., & Seshia, A. A. (2016). Acoustic biosensors. Essays in Biochemistry, 60(1), 101–110. doi: 10.1042/EBC20150011.
  Grundmann, M., & Kostenis, E. (2015a). Holistic methods for the analysis of cNMP effects. In In Handbook of experimental pharmacology. Berlin: Springer. doi: 10.1007/164_2015_42.
  Grundmann, M., & Kostenis, E. (2015b). Label‐free biosensor assays in GPCR screening. In D.M. Prazeres & S.A.M. Martins (Eds.), G protein‐coupled receptor screening assays: Methods and protocols (pp. 199–213). New York: Springer.
  Grundmann, M., Tikhonova, I. G., Hudson, B. D., Smith, N. J., Mohr, K., Ulven, T., … Kostenis, E. (2016). A molecular mechanism for sequential activation of a G protein‐coupled receptor. Cell Chemical Biology, 23(3), 392–403. doi: 10.1016/j.chembiol.2016.02.014.
  Hennen, S., Wang, H., Peters, L., Merten, N., Simon, K., Spinrath, A., … Schröder, R. (2013). Decoding signaling and function of the orphan G protein–coupled receptor GPR17 with a small‐molecule agonist. Science Signaling, 6(298), ra93. doi: 10.1126/scisignal.2004350.
  Kahsai, A. W., Wisler, J. W., Lee, J., Ahn, S., Cahill III, T. J., Dennison, S. M., … Pani, B. (2016). Conformationally selective RNA aptamers allosterically modulate the [beta] 2‐adrenoceptor. Nature Chemical Biology, 12(9), 709–716. doi: 10.1038/nchembio.2126.
  Kenakin, T. P. (2009). Cellular assays as portals to seven‐transmembrane receptor‐based drug discovery. Nature Reviews Drug Discovery, 8(8), 617–626. doi: 10.1038/nrd2838.
  Kenakin, T. (2010). A holistic view of GPCR signaling: Dynamic mass redistribution assays measure the complexity of G protein‐coupled receptor signaling. Nature Biotechnology, 28(9), 928–930. doi: 10.1038/nbt0910‐928.
  Kenakin, T. P. (2016). Synoptic pharmacology: Detecting and assessing the pharmacological significance of ligands for orphan receptors. Pharmacological Research, 114, 284–290. doi: 10.1016/j.phrs.2016.01.022.
  Kim, A. R., Ulirsch, J. C., Wilmes, S., Unal, E., Moraga, I., Karakukcu, M., … Sankaran, V. G. (2017). Functional selectivity in cytokine signaling revealed through a pathogenic EPO mutation. Cell, 168(6), 1053–1064.e1015. doi: 10.1016/j.cell.2017.02.026
  Konermann, S., Brigham, M. D., Trevino, A. E., Joung, J., Abudayyeh, O. O., Barcena, C., … Zhang, F. (2015). Genome‐scale transcriptional activation by an engineered CRISPR‐Cas9 complex. Nature. 517, 583–588.
  Leung, G., Tang, H. R., McGuinness, R., Verdonk, E., Michelotti, J. M., & Liu, V. F. (2005). Cellular dielectric spectroscopy: A label‐free technology for drug discovery. Journal of the Association for Laboratory Automation, 10(4), 258–269.
  Lieb, S., Michaelis, S., Plank, N., Bernhardt, G., Buschauer, A., & Wegener, J. (2016). Label‐free analysis of GPCR‐stimulation: The critical impact of cell adhesion. Pharmacological Research, 108, 65–74. doi: 10.1016/j.phrs.2016.04.026.
  Matano, M., Date, S., Shimokawa, M., Takano, A., Fujii, M., Ohta, Y., … Sato, T. (2015). Modeling colorectal cancer using CRISPR‐Cas9‐mediated engineering of human intestinal organoids. Nature Medicine, 21(3), 256–262. doi: 10.1038/nm.3802.
  Meyers, J., Craig, J., & Odde, D. J. (2006). Potential for control of signaling pathways via cell size and shape. Current Biology, 16(17), 1685–1693. doi: 10.1016/j.cub.2006.07.056.
  Mullard, A. (2015). The phenotypic screening pendulum swings. Nature Reviews Drug Discovery, 14(12), 807–809. doi: 10.1038/nrd4783.
  Peters, M. F., Knappenberger, K. S., Wilkins, D., Sygowski, L. A., Lazor, L. A., Liu, J., & Scott, C. W. (2007). Evaluation of cellular dielectric spectroscopy, a whole‐cell, label‐free technology for drug discovery on Gi‐coupled GPCRs. Journal of Biomolecular Screening, 12(3), 312–319. doi: 10.1177/1087057106298637.
  Peters, M. F., & Scott, C. W. (2009). Evaluating cellular impedance assays for detection of GPCR pleiotropic signaling and functional selectivity. Journal of Biomolecular Screening, 14(3), 246–255. doi: 10.1177/1087057108330115.
  Peters, M. F., Vaillancourt, F., Heroux, M., Valiquette, M., & Scott, C. W. (2010). Comparing label‐free biosensors for pharmacological screening with cell‐based functional assays. Assay and Drug Development Technologies, 8(2), 219–227. doi: 10.1089/adt.2009.0232.
  Phelan, K. and May, K. M. (2015). Basic techniques in mammalian cell tissue culture. Current Protocols in Cell Biology, 66, 1.1.1‐1.1.22. doi: 10.1002/0471143030.cb0101s66.
  Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013). Repurposing CRISPR as an RNA‐guided platform for sequence‐specific control of gene expression. Cell, 152(5), 1173–1183. doi: 10.1016/j.cell.2013.02.022.
  Sang, S., Wang, Y., Feng, Q., Wei, Y., Ji, J., & Zhang, W. (2016). Progress of new label‐free techniques for biosensors: A review. Critical Reviews in Biotechnology, 36(3), 465–481. doi: 10.3109/07388551.2014.991270.
  Scannell, J. W., Blanckley, A., Boldon, H., & Warrington, B. (2012). Diagnosing the decline in pharmaceutical R&D efficiency. Nature Reviews Drug Discovery, 11(3), 191–200. doi: 10.1038/nrd3681.
  Schrage, R., Schmitz, A.‐L., Gaffal, E., Annala, S., Kehraus, S., Wenzel, D., … Shinjo, Y. (2015). The experimental power of FR900359 to study Gq‐regulated biological processes. Nature Communications, 6, 10156. doi: 10.1038/ncomms10156.
  Schröder, R., Janssen, N., Schmidt, J., Kebig, A., Merten, N., Hennen, S., … Zahn, S. (2010). Deconvolution of complex G protein‐coupled receptor signaling in live cells using dynamic mass redistribution measurements. Nature Biotechnology, 28(9), 943–949. doi: 10.1038/nbt.1671.
  Schröder, R., Schmidt, J., Blättermann, S., Peters, L., Janssen, N., Grundmann, M., … Drewke, C. (2011). Applying label‐free dynamic mass redistribution technology to frame signaling of G protein‐coupled receptors noninvasively in living cells. Nature Protocols, 6(11), 1748–1760. doi: 10.1038/nprot.2011.386.
  Schuck, P. (1997). Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules. Annual Review of Biophysics and Biomolecular Structure, 26(1), 541–566. doi: 10.1146/annurev.biophys.26.1.541.
  Schumann, K., Lin, S., Boyer, E., Simeonov, D. R., Subramaniam, M., Gate, R. E., … Doudna, J. A. (2015). Generation of knock‐in primary human T cells using Cas9 ribonucleoproteins. Proceedings of the National Academy of Sciences, 112(33), 10437–10442. doi: 10.1073/pnas.1512503112.
  Scott, C. W., & Peters, M. F. (2010). Label‐free whole‐cell assays: Expanding the scope of GPCR screening. Drug Discovery Today, 15(17), 704–716. doi: 10.1016/j.drudis.2010.06.008.
  Shalem, O., Sanjana, N. E., Hartenian, E., Shi, X., Scott, D. A., Mikkelsen, T. S., … Doench, J. G. (2014). Genome‐scale CRISPR‐Cas9 knockout screening in human cells. Science, 343(6166), 84–87. doi: 10.1126/science.1247005.
  Sun, H., Wei, Y., Deng, H., Xiong, Q., Li, M., Lahiri, J., & Fang, Y. (2014). Label‐free cell phenotypic profiling decodes the composition and signaling of an endogenous ATP‐sensitive potassium channel. Scientific Reports, 4, 4934. doi: 10.1038/srep04934.
  Tran, E., & Fang, Y. (2009). Label‐free optical biosensor for probing integrative role of adenylyl cyclase in G protein‐coupled receptor signaling. Journal of Receptors and Signal Transduction, 29(3–4), 154–162. doi: 10.1080/10799890903052544.
  Valbuena, S., & Lerma, J. (2016). Non‐canonical signaling, the hidden life of ligand‐gated ion channels. Neuron, 92(2), 316–329. doi: 10.1016/j.neuron.2016.10.016.
  van der Greef, J., & McBurney, R. N. (2005). Rescuing drug discovery: In vivo systems pathology and systems pharmacology. Nature Reviews Drug Discovery, 4(12), 961–967. doi: 10.1038/nrd1904.
  Verdonk, E., Johnson, K., McGuinness, R., Leung, G., Chen, Y.‐W., Tang, H. R., … Liu, V. F. (2006). Cellular dielectric spectroscopy: A label‐free comprehensive platform for functional evaluation of endogenous receptors. Assay and Drug Development Technologies, 4(5), 609–619. doi: 10.1089/adt.2006.4.609.
  Verzijl, D., Riedl, T., Parren, P., & Gerritsen, A. (2017). A novel label‐free cell‐based assay technology using biolayer interferometry. Biosensors and Bioelectronics, 87, 388–395. doi: 10.1016/j.bios.2016.08.095.
  Vincent, F., Loria, P., Pregel, M., Stanton, R., Kitching, L., Nocka, K., … Schroeter, T. (2015). Developing predictive assays: The phenotypic screening “rule of 3”. Science Translational Medicine, 7(293), 293ps215–293ps215. doi: 10.1126/scitranslmed.aab1201.
  Wagner, B. K. (2016). The resurgence of phenotypic screening in drug discovery and development. Expert Opinion on Drug Discovery, 11, 121‐125.
  Wang, G., Dewilde, A. H., Zhang, J., Pal, A., Vashist, M., Bello, D., … Therrien, J. M. (2011). A living cell quartz crystal microbalance biosensor for continuous monitoring of cytotoxic responses of macrophages to single‐walled carbon nanotubes. Particle and Fibre Toxicology, 8(1), 4. doi: 10.1186/1743‐8977‐8‐4.
  Wu, M., Coblitz, B., Shikano, S., Long, S., Spieker, M., Frutos, A. G., … Li, M. (2006). Phospho‐specific recognition by 14‐3‐3 proteins and antibodies monitored by a high throughput label‐free optical biosensor. FEBS Letters, 580(24), 5681–5689. doi: 10.1016/j.febslet.2006.09.019.
  Wu, M., & Li, M. (2015). Resonant waveguide grating for monitoring biomolecular interactions. Protein‐Protein Interactions: Methods and Applications, 1278, 139–152.
  Yanase, Y., Hiragun, T., Ishii, K., Kawaguchi, T., Yanase, T., Kawai, M., … Hide, M. (2014). Surface plasmon resonance for cell‐based clinical diagnosis. Sensors, 14(3), 4948–4959. doi: 10.3390/s140304948.
  Zaytseva, N., Lynn, J. G., Wu, Q., Mudaliar, D. J., Sun, H., Kuang, P. Q., & Fang, Y. (2013). Resonant waveguide grating biosensor‐enabled label‐free and fluorescence detection of cell adhesion. Sensors and Actuators B: Chemical, 188, 1064–1072. doi: 10.1016/j.snb.2013.08.012.
  Zheng, W., Thorne, N., & McKew, J. C. (2013). Phenotypic screens as a renewed approach for drug discovery. Drug Discovery Today, 18(21), 1067–1073. doi: 10.1016/j.drudis.2013.07.001.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library