Corning Epic System Applications The Corning Epic System can be applied to probe many of the biomolecular interactions involved in cellular and molecular biology. Beyond its application to direct binding, label-free HTS assays on immobilized therapeutic targets, many assays were derived based on isolated proteins (kinases, proteases, peptides, and antibodies), membrane preparations (GPCR), cell lysates and whole cells cultured directly on the Epic microplate. This flexibility in assay design includes functional and competitive formats in order to increase the information content beyond direct binding. Furthermore, cells cultured directly on the Epic microplate were shown to respond upon addition of various stimuli. Therapeutic Application The detection of biochemical and cell-based binding interactions is critical to understanding the diseased state and identifying compounds which can disrupt undesirable interactions. Disruption of certain interactions may result in therapies within various target areas such as: Oncology Cardiology Neurology (CNS) Immunology Respiratory/Pulmonary Infectious Diseases Inflammation
Representative Biochemical and Cell-Based Assays Performed with the Epic System 2
Applications Overview Many applications have been demonstrated using the Epic System summarized in the table below: Interaction of Interest Example Assay Status/Reference BIOCHEMICAL ASSAYS Drug/Protein Drug/Kinase Direct Bind BIPI 2 presentation at SBS 4 25 conference Functional Kinase Assay JJPRD 3 / Corning ACT 7 24 conference poster Drug/Protease Direct Bind Functional Protease Assay Warfarin/HAS & Digitoxin/HAS Assay demonstrated by Corning Vancomycin/D-Ala D-Ala Corning presentation at Lab Automation 25 conference Drug/Carbonic Anhydrase Corning Miptec 8 26 conference poster Protein/Protein Kinase/Peptide Assay demonstrated by Corning Cytokine/Cytokine Receptor JJPRD 3 /Corning ACT 7 24 conference poster Cytokine/IgG Assay demonstrated by Corning JJPRD 3 /Corning ACT 7 Antiphosphotyrosine Antibody/ 24 Peptide conference poster Antibody/Antibody Corning presentation at Lab Automation 25 conference Antibody/Antigen (in serum) Multiple Cytokines JJPRD 3 /Corning ACT 7 24 conference poster Drug/Peptide D-Ala D-Ala/Vancomycin Corning presentation at Lab Automation 25 conference Protein/DNA Protein/DNA JJPRD 3 /Corning ACT 7 24 conference poster CELL-BASED ASSAYS Cell Signaling EGFR pathway exploration See 25 publication in Analyzed Chemistry 5 Cytoskeletal Disruption Modulators of cytoskeletal See 25 publication in FEBS structure based actin filament letters 6 GPCR Assays PAR 1 receptor in CHO-K1 Corning Miptec 8 26 conference poster EGFR Assays EGFR Assay in A431 See 25 publication in FEBS letters 6 Lipid Signaling Cytotoxicity Proliferation Assays Cell Adhesion HYBRID ASSAYS Cell growth rate in presence of compound Cell attachment time Cellular functions (BBA Fang) Cell Lysate Protein Detection of a specific protein Assay demonstrated by Corning Detection in cell lysate Cell Culture Supernatant Detection of a specific protein JJPRD 3 /Corning ACT 7 24 Protein Detection in cell culture supernatant conference poster 1 Interaction of interest is shown as analyte followed by immobilized target (ie: Drug-Protein). 2 BIPI: Boehringer-Ingelheim Pharmaceuticals Incorporated, Ridgefield, CT. 3 JJPRD: Johnson and Johnson Pharmaceutical Research and Development, Raritan, NJ. 4 SBS: Society for Biomolecular Screening, Geneva, Switzerland September 25. 5 Characteristics of Dynamic Mass Redistribution of EGF Receptor Signaling in Living Cells Measured with Label-Free Optical Biosensors. Fang, Y., Ferrie, A., Fontaine, N., Yuen, P. (Sept 25). Analytical Chemistry. 6 Probing Cytoskeleton Modulation by Optical Biosensors. Fang, Y., Ferrie, A., Guangshan, L. (Aug 25). FEBS Letters 579, 4175-418. 7 IBC Life Sciences Assays and Cellular Targets, San Diego, CA October 24. 8 MipTec Conference, Basel, Switzerland, May 26. 3
Biochemical Assays Example Drug/Protein Assay: Drug/Kinase (Direct Bind) Kinase activity is of tremendous interest in disease areas including cancer, inflammation, and obesity. Drugs which bind and interact with kinases are therefore of great interest to the drug discovery community. Drugs which directly bind kinases can be detected on the Epic System. In the following example a kinase is immobilized directly to the microplate and exposed to various binding and nonbinding drugs during analyte addition. Binding of a drug to the kinase can be detected by a shift in resonant wavelength. Method In this experiment, a 4 kda kinase was immobilized to the surface of the Epic microplate. Various drugs (both known binders and non-binders which are ~3 Da) were added to their respective wells on the standard 384 well Epic microplate. Response (pm) 14 12 1 8 6 4 2-2 -4-6 Drug Binding Immobilized Kinase Binder Non-binder Binding of drug to the kinase target is shown below. Note that drugs which bind generally show a shift of ~1 pm over drugs which do not bind to the kinase target. Kinase target Kinase target Drug addition step Drug addition step Drug Drug Binding Drug Non-binding drug 4
Biochemical Assays Example Protein/Protein Assay: Kinase/Peptide The binding of kinases to peptides is critical in the activation of such peptides via phosphorylation in signal cascades. Determining methods to disrupt this kinase peptide interaction is of interest to therapeutic groups studying the diseased state. The purpose of this experiment was to determine if kinase-peptide binding interaction could be detected on the Epic System. Methods Peptide (1.2 kda) recognized by the kinase was immobilized to the Epic microplate. Negative controls were prepared using streptavidin which does not recognize the peptide. After immobilization of the same peptide to all wells, kinase was added to certain wells, while streptavidin was added to negative control wells as shown in the diagram below. Kinase added to wells during addition step Streptavidin added to wells during addition step Kinase binds peptide Streptavidin does not bind The Epic System was clearly able to detect the binding of the kinase to the peptide. Binding resulted in a shift of ~23 pm relative to wells where streptavidin did not bind to the peptide. This experimental model may be useful in determining the inhibitory effects of compounds in kinase-peptide interaction pathway exploration where the peptide substrate for a kinase is promiscuous or unknown. The resulting binding signal in this assay was lower than anticipated given the size Drug Binding Immobilized Peptide 4 of the analyte (~45 kda) with respect to the 35 immobilized target (~1.2 kda). This low 3 binding signal may be attributed the lysine rich binding region on the peptide. These 25 lysine residues are involved in immobilization. Immobilization at these lysine regions 15 2 may render the peptide inaccessible to kinase 1 binding in some cases. Therefore, additional 5 immobilization techniques (i.e., affinity capture) may be explored to produce higher Kinase Sv (neg. control) binding signals. Such methods may provide better binding microenvironment. Response (pm) 5
Biochemical Assays Example Drug/Protein Assay: Kinase Inhibition with Drug (functional assay) In this assay, the Epic System was tested using a simple kinase functional model demonstrating the feasibility of running a label-free, functional kinase assay. Binding would be dependent on phosphorylation of the substrate and therefore dependent on kinase activity. Methods The peptide substrate was immobilized on the Epic microplate then reacted with kinase and ATP in the presence and absence of a proprietary Johnson and Johnson Pharmaceutical Research and Development inhibitor compounds (JNJX) as shown in the diagram below. ATP Peptide Drug No Drug Cytokine Receptor/Fc Complex Drug Prevents Peptide Phosphorylation Kinase Phosphorylates Peptide Epic Microplate Titration of JNJX drug compound indicates a dose-dependent shift in resonance wavelength when compared to positive control (left). The IC 5 curve for JNJX (right) is also shown. Resonant Shift (pm) 12 1 8 6 4 2-2 2 2.2.2.2 Compound Dose (µm) No drug JNJX Percent Inhibition 1 8 6 4 IC 5 = 1.7e-7 2-11 -1-9 -8-7 -6-5 -4-3 JNJX (M) Functional Kinase Activity Inhibited by Drug Compounds 6 These results suggest that the reaction of peptide target to the kinase was inhibited in the presence of the drugs tested. Furthermore, the inhibition occurred as the drug specifically bound to the kinase, not the peptide, as the kinase was pre-incubated with drugs testes. The inhibition of the kinase resulted in failure of the kinase to phosphorylate the substrate. As a result the antiphosphotyrosine antibody could not recognize the unphosphorylated target and failed to indicate binding. Inhibition was dose dependent; as more drug was present, the inhibitory effect on kinase activity was more pronounced.
Biochemical Assays Example Protein/Protein Assay: Cytokine Receptor/Cytokine Cytokines are among the most studied therapeutic targets in drug discovery. Interactions between cytokines and various receptors have been known to be a critical factor in various disease states including oncogenesis and inflammation. In the following assay, the Epic System was used to detect the binding of an immobilized cytokine to its receptor. These assays are courtesy of Johnson and Johnson Pharmaceutical Research and Development, Raritan, NJ. Methods Cytokine was immobilized to the Epic microplate. Immobilization conditions were optimized by monitoring the immobilization process on the Epic System. After immobilization, the target cytokine was exposed to its own receptor. A subset of receptors was pre-incubated with cytokine at various concentrations to determine specificity of binding interaction as shown in the diagram below. Cytokine Receptor Complex Assay 1: Receptor Binding Assay 2: Inhibition Assay The addition of cytokine receptor to the immobilized cytokine target resulted in shift of ~45 pm. To determine if the response was due to specific binding of the cytokine receptor to the immobilized target, cytokine was pre-incubated with the receptor to determine if the binding interaction could be inhibited. The results indicated the dose dependent inhibition of cytokine-cytokine receptor binding interaction when the receptor was pre-incubated with cytokine. Further studies to identify drug compounds which may interfere with this interaction would be of interest to various therapeutic areas in drug discovery. 5 5 Response (pm) 4 3 2 1 4 3 2 1 5 1 15 2 5 1 15 2 [cytokine receptor] (nm) Preincubation with Cytokine (µg/ml) 7
Biochemical Assays Example Drug/Protein Assay: Drug on Carbonic Anhydrase Detection of small molecule compounds binding to large protein targets is of great interest in label-free technologies. To determine if a ~3 Da small molecule can be detected binding to a large protein target, the following experiment was conducted. Methods Carbonic anhydrase was immobilized to the Epic microplate. A known binding drug furosemide (331 Da) was added at various concentrations during the single analyte addition step. The results were recorded and plotted as concentration furosemide (µm) against corrected response (pm). The diagram to the right illustrates the direct binding experimental plan. Binding of furosemide is dose dependent and saturable. Estimated KD is in good agreement with literature. Given that it is possible to detect this interaction, the Epic System should be explored further for direct drug screening on immobilized protein target. Furosemide Addition Furosemide binds carbonic anhydrase 4.5 4. 3.5 Response (pm) 3. 2.5 2. 1.5 K 1. D = 49 nm R 2 =.9.5 2.5 5. 7.5 1. 12.5 [furosemide] (µm) Cell-Based Assays Example Cytoskeletal Disruption Assay: Cytoskeleton Modulation The cytoskeleton is a complex and dynamic network of protein filaments that extends throughout the cytoplasm of eukaryotic cells. Conventional methods for probing cytoskeleton structure mainly utilize the staining of cells with fluorescently labeled molecules that specifically bind to certain cytoskeleton components such as F-actin or tubulin. We used the Epic System to monitor in real time the action of saponin a plant-derived glycoside that is well known to disrupt the cell plasma membranes and causes the leakage of the intracellular components on living cells. 6
Methods Chinese hamster ovary (CHO) cells were cultured in a 96-well Epic microplate. Prior to an assay, cells were washed with a solution of Hanks balanced salt solution (HBSS) and then maintained in 1 µl of HBSS. Cells were pretreated by adding 5 µl HBSS followed by an addition of 5 µl of saponin. The action of saponin (75 µg/ml or 125 µg/ml) on the cells in the absence and presence of cytoskeleton modulators was monitored. The addition of saponin to cells cultured on the Epic sensor, caused a decrease in sensor response, due to permeabilization of the cell membrane and loss of intracellular material. The loss of material was dependent upon the amount of saponin that was added. SDS- PAGE was used to confirm that the decrease in sensor response was due to permeabilization of the cell membrane and loss of intracellular material. After the Epic assay was performed, the supernatant from three replicate wells was collected and run on an SDS- PAGE gel, and confirmed the presence of proteins in the supernatant (data not shown). When cells were co-incubated with saponin and one of two different compounds known to disrupt actin filaments, an increase in the loss of cellular material was observed. Specifically, cytochalasin B and latrunculin A produced an ~5% increase in the loss of material relative to incubation with saponin alone. Cytochalasin B and latrunculin A are known for their ability to cause actin filament disassembly. Coincubation of the cells with saponin and either phalloidin, vinblastine or nocodazole showed no effect relative to incubation with saponin alone. Phalloidin is a compound that promotes actin polymerization; vinblastine and nocodazole are antimitotic agents that inhibit microtubule assembly. These results demonstrate that the Epic System can be used in a whole cell assay to screen for compounds which disrupt actin filaments. 2.5.5 2. HBSS saponin Response (unit) 1.5 1..5 Response (unit) -.5 -.5 1 2 3 4 [saponin] (µg/ml) -1. 2 4 6 8 1 Time (sec) 4. Response (unit) 3. 2. 1. 75 µg/ml 125 µg/ml. HBSS phalloidin vinblastine nocodazole Cytochalasin B Compound Latrunculin A 7
Cell-Based Assays Example Cell Signaling Assay: EGFR Pathway Exploration The combinatorial integration of signaling pathways mediated through a specific molecule in response to stimuli plays an important role in the specificity of cellular responses and cell functions, as best exampled by the signaling of epidermal growth factor receptor (EGFR). Upon ligand binding, the EGFR becomes dimerized and activated through the autophosphorylation of the receptor on tyrosine residues in the cytoplasmic domain, thus initiating a number of intracellular signals by interacting with distinct signaling proteins. The Ras/MAPK pathway mediated through EGFR is well known to proceed via the signaling proteins Shc, Grb2, Sos, Ras, Raf, MEK, ERK and ERK/MAPK. The MAPK pathway regulates a diverse array of cellular functions including cell proliferation, survival, motility, differentiation, and cycling. However, the specificity of cell responses is largely determined by the integration of signaling network interactions, and depends on the cellular context. Traditional cell-based methods rely on the measurements of a single cellular event, such as second messenger generation (e.g., Ca2+ flux, camp level changes), or the translocation of a particular target tagged with a fluorescence label, or the expression of a reporter gene. These methods can provide functional, kinetic cell-based information on the cellular consequences of targetcompound interaction, including drug mechanisms of action, efficacy, selectivity and cytotoxicity. However, these methods tend to fail to generate information relate to the overall cellular responses, because of the complexity of cell function as well as the dependence of specific cell responses on the integration of multitude signaling pathways. The Epic System was used to monitor the EGF-induced dynamic mass redistribution in human epidermoid carcinoma A431 cells, and for the first time studied the signaling networks that contribute to the mass redistribution signal observed. Methods Human epidermoid carcinoma A431 cells (a cell line with endogenously overexpressed EGFRs) and Chinese hamster ovary cells (a cell line that does not express EGFRs), were cultured in a 96-well Epic microplate. Two sets of experiments were performed. The dose dependent response of the cells to EGF was studied by titrating the amount of epidermal growth factor (EGF) added to each well. In a second set of experiments, the role of EGFR phosphorylation on the overall directional mass redistribution (DMR) response was investigated, by preincubating the cells with a potent and specific EGFR kinase inhibitor, AG1478. Upon EGF stimulation, there are many events that lead to mass redistribution in A431 cells. For example, EGFR activation leads to the recruitment of intracellular components to the activated receptor, the movement of the resulted complexes, and the rearrangement of cytoskeleton structure that ultimately results in chemokinetic motility. When such movements or changes occur in the cell near the sensor surface, a DMR signal is generated, that is an integrated signature for EGFR activation. Experiments showed that the magnitude of the DMR signal was dependent on the amount of EGF added. As a control, no response was observed when EGF was added to CHO cells cultured on the sensor surface. CHO cells do not express EGFRs. Pretreatment of the cells with AG1478, a potent and specific EGFR kinase inhibitor, resulted in a dose dependent suppression of the EGF response with an IC5 of ~194 nm. Taken together, these data suggest that the Epic System can be used to perform whole cell assays to screen for inhibitors of the EGF receptor. 1
Response (N-DMR) (unit) 3 2 1 Response (unit) -1 1 2 3 4-9. -6.5-4. [EGF] (nm) log AG1478] (M) 3 2 1 Hybrid Assays Example Pathway Exploration: Protein Detection in Whole Cell Lysate The industry s knowledge of molecular interactions is limited to a small subset of known molecular entities in the whole cell. Furthermore, identification, isolation, and overexpression of proteins of interest is both labor intensive and may be cost prohibitive. The purpose of the following experiment was to determine if protein expression in a whole cell lysate can be determined without the purification or isolation. Methods Cells were induced to express high levels of a cytokine of interest. Antibodies to the cytokine of interest were then immobilized on the Epic microplate. Cell lysate was added to each well. In negative control wells the cytokine of interest was immunoprecipitated to remove the protein as shown in the diagram below. After lysate addition, washing steps removed a significant amount of non-specifically attached materials. Lysate containing cytokine Lysate cytokine immunoprecipitated Analyte addition step Cytokine binds Ab No cytokine binding 11
After the antibody to the cytokine was immobilized to the Epic Microplate, lysate was added to each well. Addition of lysate resulted in a large bulk index shift in excess of 1nm. Washing steps significantly reduced the bulk index shift and removed non-specifically attached materials. The lysate containing cytokine showed a greater resonant shift relative to the same lysate that had been previously immunoprecipitated to remove the cytokine being tested. 37 Response (pm) 36 35 34 33 Cell Lysate + cytokine Cell lysate cytokine 32 31 N = 4, cv = 18% For additional product or technical information, please visit www.corning.com/ lifesciences/epic, or call 8.492.111, or e-mail at epic@corning.com. Customers outside the United States, please call +1.978.442.22 or contact your local Corning sales office listed below. Corning Incorporated Life Sciences Tower 2, 4th Floor 9 Chelmsford St. Lowell, MA 1851 t 8.492.111 t 978.442.22 f 978.442.2476 www.corning.com/lifesciences Worldwide Support Offices ASIA/PACIFIC Australia t 61 2-9416-492 f 61 2-9416-493 China t 86 21-3222-4666 f 86 21-6288-1575 Hong Kong t 852-287-2723 f 852-287-2152 India t 91-124-235 785 f 91-124-41 27 Japan t 81 () 3-3586 1996/1997 f 81 () 3-3586 1291/1292 Korea t 82 2-796-95 f 82 2-796-93 Singapore t 65 6733-6511 f 65 6861-2913 Corning and Epic are registered trademarks of Corning Incorporated, Corning, NY. Corning Incorporated, One Riverfront Plaza, Corning, NY 14831-1 Taiwan t 886 2-2716-338 f 886 2-2716-339 EUROPE France t 8 916 882 f 8 918 636 Germany t 8 11 1153 f 8 11 2427 The Netherlands t 31 2 655 79 28 f 31 2 659 76 73 United Kingdom t 8 376 866 f 8 279 1117 All Other European Countries t 31 () 2 659 6 51 f 31 () 2 659 76 73 LATIN AMERICA Brasil t (55-11) 389-7419 f (55-11) 3167-7 Mexico t (52-81) 8158-84 f (52-81) 8313-8589 27 Corning Incorporated Printed in USA 3/7 CLS-AN-73REV1