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High-Throughput Cardiotoxicity Screening Assays with the BIND Label-Free PlatformAnimal models are frequently used to test compounds for in vivo toxicity during preclinical drug development. These models, while useful, are expensive and low-throughput. For this reason, cell-based assays are increasingly being used to test compounds for cytotoxicity prior to advancing to animal models. A universal cell-based assay that could predict in vivo toxicity of compounds would reduce the cost and the timeline of preclinical compound development by allowing earlier go/no go decisions. SRU Biosystems has developed a new label-free, high-throughput toxicity application that provides the opportunity for multiple high value readouts in a single assay. The system has been developed for 1536-well screening and has direct application for use with stem cell-derived cardiomyocytes and hepatocytes. Toxins that work through distinct mechanisms of action (e.g. DNA damaging agents, protein synthesis inhibitors, microtubule inhibitors) generate label-free kinetic profiles on BIND that serve as signatures for that particular cell death mechanism. Furthermore, ES-derived cardiomyocytes can be readily grown on BIND® Biosensor plates and display a healthy, beating phenotype after a couple of days in culture. With rapid (up to 4/second) label-free measurements enabled, the BIND® Reader displays a beating phenotype as real-time oscillations of positive-to-negative PWV shifts that accurately report on beating rate as confirmed by bright-field microscopy. Thus, in addition to cell death and mechanism of action, BIND toxicity assays with cardiomyocytes can also include readouts on beating frequency and amplitude and will serve as high value, early stage in vitro assays for the filtering of drugs with cardiotoxic qualities. The BIND system, therefore, offers a high-throughput universal system for detecting compound cytotoxicity that allows predictions of compound mechanism to be made.>> Download PDF |
Chemotaxis in High-Throughput: Label-Free and Transwell-Free BIND AssaysCell migration in response to environmental stimuli is central to a broad range of physiological processes, including immune responses, wound healing, and stem cell homing. In some cases, excessive cell migration can contribute to disease pathologies, including inflammatory diseases and tumor metastasis. Drug discovery efforts for inhibitors of cell migration are hampered by the lack of high throughput assays to enable primary screening campaigns in functionally relevant cell types. SRU Biosystems has developed a label-free, high resolution BIND SCANNER using our optical resonance detection technology that has enabled the implementation of high throughput screening assays for chemotaxis. The BIND “Touchdown” assay measures the migration of cells through an artificial basement membrane and onto the biosensor surface from which a gradient of chemokine is released. The “Lift-Off ” assay measures the detachment of cells away from the collagen-coated sensor surface toward chemokine presented in the bath media. Both assay s are independent of transwells and can be run in 96-, 384- or 1536-well formats. Furthermore, the software masking algorithms being developed for the high resolution Scanner allow for low cell density migration events to be accurately quantified. The result are chemotaxis assays that can be implemented with cell populations that are limited in supply, such as primary cultured cells. Using these assays, we have demonstrated chemotaxis of mesenchymal stem cells in response to specific chemokines and have validated the assays with neutralizing antibodies to both chemokines and their cell surface receptors. Additional chemotaxis assays in development include a transmigration assay in which the migration of cells can be measured across an endothelial cell layer onto a chemokine-coated biosensor surface. SRU Biosystem’s high-throughput BIND chemotaxis assays will enable primary screening for important modulators of cell migration in functionally relevant cell types.>> Download PDF |
High Throughput, Label-free BIND Assays for Monitoring Stem Cell Differentiation.SRU Biosystems’ optical, label-free BIND® technology provides a quantitative method for measuring cellular responses that involve changes in cell morphology or adherence to extracellular matrix (ECM)-coated biosensors. Mesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal that can differentiate into several cell types, including osteoblasts, chondrocytes and adipocytes. MSCs have demonstrated significant clinical potential and yet high-throughput approaches for screening compounds that affect MSC differentiation are limited. We have developed the BIND® SCANNER, a label-free, high resolution instrument, using our optical resonance detection technology that has enabled the implementation of high throughput assays for monitoring MSC differentiation into bone-producing osteoblasts. MSCs can be readily propagated on extracellular matrix-coated, 384-well optical biosensors. Following addition of a differentiation cocktail, MSCs fully differentiate into bone-producing osteoblasts on BIND® Biosensors as measured by standard staining reagents and biochemical analyses. Daily measurements taken on the high resolution BIND SCANNER reveal significant shifts (>30nm) in Peak Wavelength Value (PWV) as differentiating osteoblasts secrete collagen and mineralized deposits onto the sensor surface. These PWV shifts can be quantified and expressed as a differentiation time course – all from a single well of a 384-well biosensor plate. The BIND readout is more sensitive than traditional mineralization staining as the PWV shifts precede Alizarin Red staining by 3-4 days. Moreover, small molecules that modulate the time course of MSC-osteoblast differentiation can be easily detected. Thus, SRU’s label-free stem cell assays enable high-throughput phenotypic screens of small molecule libraries for hit identification to further elucidate the molecular targets involved in stem cell differentiation.>> Download PDF |
From Engineered Cell Lines to Stem Cells: A High-Throughput, Label-Free Assay for SDF-1/CXCR4 Signaling.Stromal cell-derived factor-1 (SDF-1) is a chemokine that binds to and activates the G-protein coupled receptor, CXCR4. The SDF-1/CXCR4 signaling axis plays a critical role in the homing of stem and progenitor cells during embryogenesis and organ regeneration and of lymphocytes to sites of inflammation. SDF-1 also contributes to the metastasis of CXCR4-expressing tumor cells and is an important target for drug discovery efforts in multiple disease indications. SRU Biosystems’ optical, label-free BIND technology provides a quantitative method for measuring cellular responses that involve changes in cell morphology or adherence to the extracellular matrix (ECM). As acute stimulation of cells with SDF-1 induces significant restructuring of the actin cytoskeleton we sought to develop a cell-based assay on BIND ECM-coated biosensors as a readout for SDF-1/CXCR4 signaling. Cells that recombinantly express CXCR4 respond to SDF-1 with a robust, dose-dependent signal as measured with our label-free BIND technology. Real-time data collection indicates that the temporal response to SDF-1 and the concentration dependency is comparable to that observed in orthogonal assays. We have also been highly successful in measuring strong SDF-1-induced responses in several suspension cell lines expressing endogenous levels of CXCR4, including Jurkat, CEM, and Thp-1 cells. The cellular response elicited by SDF-1 on BIND biosensors is mediated by signaling through CXCR4 as neutralizing antibodies and small molecule inhibitors of CXCR4 block the response. Mesenchymal stem cells (MSCs) respond to gradients of SDF-1 by homing towards targeted areas in vivo. We have also identified conditions to readily propagate MSCs on our biosensors and show that they generate robust responses to acute stimulation with SDF-1. The data presented here demonstrate that the BIND label-free system is highly suitable as a screening assay for the SDF-1/CXCR4 signaling axis and likely to extend to other members of the chemokine/chemokine receptor family.>> Download PDF |
High Throughput, Label-free Assays for Monitoring Stem Cell Migration and DifferentiationMesenchymal stem cells (MSCs) possess significant clinical potential as multipotent cells capable of selfrenewal that can differentiate into several cell types, including osteoblasts, chondrocytes and adipocytes. SRU has developed label-free assays using our optical resonance detection technology to enable high throughput screening of MSC migration and differentiation. MSCs can be readily propagated on extracellular matrixcoated optical biosensors and respond to a bath application of chemokines with robust, dose-dependent, and highly sensitive label-free responses. The “Lift-Off ” cell migration assay measures the detachment of cells away from the collagen-coated sensor surface toward chemokine presented in the bath media. The assay is independent of transwells, requires low cell numbers per well, and is 1536-well compatible. Our label-free assay for measuring MSC-osteoblast differentiation is characterized by unique label-free signals as mineral deposits are formed on the sensor surface. The real-time readout displays complete differentiation phenotypes in a single well, is more sensitive than traditional staining reagents, and can be applied in high-throughput for screening small molecule or RNAi libraries to monitor increases or decreases in the rate of differentiation or self-renewal.>> Download PDF |
A Label-free Cell-based Functional Assay for Identification and Characterization of TRPV1 Channel Modulators.The vaniloid receptor TRPV1 is a nonselective cation channel activated by capsaicin, low pH and high temperature.Modulation of TRPV1 function provides a potentially novel approach to treatment of a variety of disorders, such as chronic pain and inflammation.To meet drug discovery need, we developed a label-free cell-based assay for identification and characterization of TRPV1 modulators utilizing the BIND®technology from SRU Biosystems Inc.The SRU BIND®technology employs photonic crystal biosensors to monitor cellular responses.Activation of TRPV1 channel triggers a robust morphological change on the sensor, which is quantifiable through the measurement of the shift of the peak wavelength.The assay is highly sensitive, allowing detection of the weak response induced by the putative natural activator anandamide and NADA, in addition to the strong response stimulated by capsaicin or resiniferatoxin.The morphological response mediated by TRPV1 is dependent on calcium influx and inhibited by staurosporin and cytochalasin D.Utilizing the new label-free cell-based assay, we characterized a panel of known activators and inhibitors of TRPV1 channel. The EC50, IC50and efficacy values derived from the new assay were in general agreement with literature report.With the new assay, we have uncovered different modes of action for different classesof TRPV1 inhibitors.Furthermore, we evaluated this new assay in a HTS environment and analyzed the correlation with other assay formats, including FLIPR with calcium dye, voltage-sensitive dye, atomic absorption and IonworkTMelectrophysiology.We conclude that the label-free cell-based functional assay is a valuable tool for discovery and characterization of new TRPV1 modulators.>> Download PDF |
A protein domain-peptide interaction study using the SRU BIND™ technologyThe biochemical target that we are interested in is the indirect blockage of the up-regulation of a growth factor important in modulating the progression of breast and prostate cancer by antagonizing the interaction of the PDZ domain of one protein with the C-terminal end of another protein that are co-regulators of the growth factor expression levels. Having no prior knowledge of the strength of the protein-peptide interaction of interest to us as a potential target for the detection of antagonists of the interaction that could be useful as therapeutics, we decided to investigate the label-free SRU BIND technology for proof-of-principle for hypothesis that we could a) detect binding between peptides and the PDZ domain of the target protein; b) quantify the interaction between peptides and the protein and c) determine if free peptides can inhibit the protein-peptide interaction.>> Download PDF |
Label-free Assays Lead HTS in New Directions. Nuclear Receptor-Small Molecule Screen Identifies New Chemical ToolsNuclear receptors are therapeutic targets of high interest and value for pharmaceutical companies. Optical label-free technology based on resonant grating sensors provides a quantitative method for directly measuring binding of small molecules to immobilized protein targets in a microplate format. This approach applies miniaturization, nanotechnology, and advanced optoelectronics technology to the detection of a biological event without the need to label either the small molecules or the protein targets. We used this technology to screen small molecule compounds against an orphan nuclear receptor and to achieve a better understanding about the biology, mechanism of action, and stoichiometry of the binding events. We present the assay development strategy and the results from a focussed screen that successfully identified small molecules as putative ligands. These ligands have been also characterised in a cellular mechanistic assay using a different technology.>> Download PDF |
Automation of SRU Biosystems’ Label-free BIND® Technology Using Beckman’s Biomek® Platform: A Universal Solution for 384- and 1536-well GPCR Screening and Profiling.Today’s drug discovery efforts require technologies that produce physically relevant data in a high throughput, cost effective manner. SRU Biosystems’ label-free BIND® technology provides the application flexibility, robust performance, and automation simplicity to meet those needs in a high density, miniaturized format. SRU had created a custom, plug-and-play platform in which the BIND ultra-high throughput reader, the SCREENER, is fully integrated within Beckman’s Biomek® FX robot for streamlined screening and profiling. This poster highlights the BIND SCREENER-Biomek FX integration platform and discusses platform components, assay performance and throughput capabilities using a cell-based GPCR assay.>> Download PDF |
 Identification of Chemical Probes for the Study of Platelet Granule SecretionThe NIH Molecular Libraries Probe Centers Network (MLPCN) initiative supports the testing of a publicly available 300K compound library to identify small molecule probes effective at modulating a given biological process or disease state. As a network member, The Broad Institute Probe Development Center (BIPDeC) participates in a comprehensive program that supports projects through assay development, execution of primary HTS, confirmation in secondary assays, and lead optimization with medicinal chemistry to identify biological probes. All data from completed probe development campaigns are made available to the wider community through PubChem. As part of this program, we undertook a screen to identify novel probes that inhibit platelet activation. Such probes will provide additional insights into the mechanisms regulating platelet granule secretion, and may also result in a regulator(s) of platelet-mediated arterial thrombosis. The screen was conducted entirely in platelet-rich plasma (PRP) obtained from normal, healthy adult donors. The screen assessed the ability of compounds to inhibit the activation of platelets when stimulated with the recombinant thrombin peptide fragment SFLLRN. To assess platelet activation, a luciferin/luciferase reaction was used to detect ATP that is released upon granule secretion. In the primary screen, the criterion for a hit was set at 50% or greater inhibition relative to the positive control Cilostazol, a known inhibitor of platelet activation. The screen showed a hit-rate of 0.2%. A ‘cherry-pick’ list of 1684 compounds was selected including all hits from the primary screen plus non-active compounds of related chemical structures. These compounds were retested in an 8-point dose response. Further secondary screens were conducted to identify false positives and select for specificity toward platelet response pathways.>> Download PDF |
 Analyzing the effects of Fibronectin Splice Variants on Growth Factor SignalingExpression of the alternatively spliced variants of fibronectin (FN) containing the EIIIA and EIIIB exons is tightly regulated during development in a tissue-specific and temporal fashion. Inclusion of these segments is seen during embryonic development of blood vessels as well as under pathological conditions in adult animals. Normally downregulated around quiescent adult vessels, EIIIA- and EIIIB-containing FNs are upregulated during wound healing, fibrosis and tumorigenesis, particularly around blood vessels. Although alternative splicing of FN is well documented, we still don't understand the molecular functions of the EIIIA and EIIIB segments. Simultaneous deletion of the EIIIA and the EIIIB exons in mice leads to embryonic lethality due to multiple defects in cardiovascular development. One striking aspect of these phenotypes is the apparent defect in proper apposition between the vascular smooth muscle cells and the endothelial cells of the dorsal aorta. This observation suggests that at least part of the vascular remodeling defects could be due to lack of proper communication between these two cell-types normally mediated through EIIIA/B containing FN. Matrix composition can affect cell signaling either through direct interactions with signaling molecules and/or their receptors, or through interactions with adhesion receptors such as integrins. To better understand the potential roles for EIIIA/B containing FN isoforms on modulation of growth factor signaling, we have been studying the responses of various cell types to stimulation with a number of growth factors when adhered to EIIIA/B containing or lacking recombinant FN fragments. Result of our studies identified differentially activated signaling pathways depending on the FN matrix provided to the cells. We are currently working on identifying individual molecules responsible in mediating differential signal transduction mediated by FN isoforms.>> Download PDF |
 Modulation of Overexpressed and Endogenous Ion Channels Measured by BIND®Ion channels are key proteins in all neurons and other CNS related cell lines. Their immense potential as drug targets is recognized by the pharmaceutical industry. The well established methods used to measure ion channel functions and to detect the compounds that modulate them - radio-ligand binding assays radioactive flux assays fluorescence-ligand binding assays, radioactive flux assays, fluorescence based assays and electro physiological methods – rely mostly on engineered cell lines, over-expressing the proteins of interest. The over-expressed ion channels do not always behave like native proteins in their native signaling context, leading to the missed prioritization of compounds. BIND® label-free universal assay platform enables detection of drug-target interactions, and provides label-free analysis of live cell target activation for both adherent and suspension cell types. In this study, we show that BIND® can monitor and measure ion channel activation as well as its specific inhibition. Ion channel activation shows clear dose dependency signals on pygnative and primary cell lines and on engineered cells, confirming the more native-like behavior of the engineered systems. This label free system has therefore the potential to support the current trend in pharmaceutical research to utilize more physiologically relevant assays earlier in the discovery process, using endogenous expression of target receptors in human primary cells for lead profiling and screening.>> Download PDF |
 The BIND™ System : Secondary Screening of Antibodies based on Antigen Specificity, Competition and Domain AnalysisOne of the challenges of using phage display or hybridoma technologies to discover therapeutic or diagnostic antibodies is the large number of antibodies that bind the antigen (soluble or on cells) in standard assays such as ELISA. The second challenge is differentiating positive antigen bindersfrom one another. The BIND™ system can be used to rank antibodies by the following criteria: relative affinity, specificity for antigen, binding to different epitopes, and competition for epitopes on soluble and cellular antigens. Data demonstrating this application will be presented. The BIND™ System, comprised of the BIND Reader™ and the BIND™ Biosensor, enables label-free measurement of interactions between biological macromolecules, small molecules, or cells in 96 and 384-well format.>> Download PDF |
 The BIND™ System : Ranking of Antibodies Early During the Screening of Hybridoma ClonesOne of the challenges of using hybridoma technologies is the large number of antibodies that bind the antigen (soluble oron cells) in standard assays such as ELISA. In order to move candidates forward in a work flow more information is required. Ranking of IgGs earlier in the process will shorten time lines for identification of lead candidates for preclinical trials. The BIND™ system allows the ranking of IgGs in HAT medium (20% bovine serum) in 3 hrs.If the assay is performed in 384 well plates, the subclass of the antibody can be determined simultaneously with ranking. Data demonstrating this application will be presented. The BIND™ System, comprised of the BIND Reader™ and the BIND Biosensor™, enables label-free measurement of interactions between biological macromolecules, small molecules, or cells in 96 and 384-well format.>> Download PDF |
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