Neuronal Screening
11:30-11:55 High-Content Fluorescent Microscopy Imaging
Approaches to Measure Rescue of Neurodegenerative Primary Neurons in a Drug Discovery Platform
Oscar Joseph Trask, Jr., Ph.D., Associate in Research, Neurobiology, Duke University
Highly complex primary co-culture systems can recapitulate more fully the complex milieu of brain tissue by supporting the interconnected growth of multiple neuronal and glial cell types. High-content imaging technology offers the promise to quantify altered morphological and functional features of individual cells in a complex heterogeneous population. We will discuss the multiple challenges encountered in the establishment of a validated model to identity and advance small molecule compounds that rescue cortical and striatial neurons from neurodegeneration in Huntington Disease. In this model, primary rat cortical and striatial neurons were transfected using electroporation to introduce truncated mutant huntingtin (htt) gene and one of many fluorescent proteins constructs (CFP, YFP, mCherry). Transfected cells were plated on supporting matrix of glia cells and treated with compounds for several days. The number of surviving neurons was determined using high-content analysis (HCA), moreover, the utility of HCA provides a means to measure dendritic morphological and the capability to measure alternative end points to better understand program cell death and cell signaling.
11:55-12:20 HCA of Primary Neurons to Identify Kinases and
Phosphatases Regulating Neurite Outgrowth
John Bixby, Ph.D., Professor, Miami Project to Cure Paralysis, University of Miami Miller School of Medicine
Recovery of function after CNS injury requires regeneration of axons and dendrites. The final effectors for these events largely comprise networks regulated by protein phosphorylation. To identify the enzymes responsible, we conducted an overexpression screen of 724 cDNAs encoding 300 protein kinases and phosphatases. Hippocampal neurons were transfected to overexpress individual kinases and phosphatases, and parameters of neurite outgrowth were measured using the Cellomics KineticScan Reader and the Neuronal Profiler Bioapplication. Surprisingly, most cDNAs did not substantially alter the length, distribution, or branching of neurites. However, we identified 77 cDNAs encoding 73 distinct kinases/phosphatases that altered one or more growth parameters. Current efforts are focused on bioinformatics approaches to understanding the signaling pathways controlled by our “hit” genes, and the degree to which distinct neuronal growth phenotypes can be recognized and classified.
 12:20-12:35 Sponsored Presentation
Analysis of Neurite Outgrowth and Neuronal
Injury Using High-Content Screening
Stella Redpath, Ph.D., Group Product Manager, in vitro
Toxicology, Millipore Corporation
Neurite formation and neuronal regeneration hold therapeutic promise for conditions such as Alzheimer’s and Parkinson’s diseases, and other CNS injuries. HCS enables evaluation of several neuronal biomarkers in a single assay. We have developed a method for large-scale, quantitative neurite outgrowth assays, and morphological screening of multiple parameters in individual cells, such as cell body count, neurite count and neurite length. The talk will focus on: validation of the assay on different cell types and co-culture conditions; evaluation of biomarkers of neurotoxicity; evaluation of high-quality immunodetection reagents for sensitivity and specificity; and assessment of promoters and inhibitors of neurite outgrowth.
12:35-1:00 Measuring Abeta-Induced Changes of Neuronal Morphology and Biochemistry in Primary Neuronal Cultures
Michael P. Bova, Ph.D., Director of Target Advancement, Elan Pharmaceuticals, Inc.
In this study, we measured Abeta (1-40) induced changes of neuronal morphology in primary rat cortical and hippocampal cultures using high-content analysis with the Cellomics neuronal profiling bioapplications. We observed that Abeta induced a dose-dependent decrease in neurite length, neurite area and branch points in primary neuronal cultures compared to non-treated cultures. In addition, we are currently investigating the effects of Abeta on markers of presynaptic function such as synapsin and synaptophysin. The conclusions of these studies will lead to a better understanding of the in vitro mechanisms of Abeta-induced cytotoxicity.
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Label-Free Screening
11:30-11:55 Use of Label-Free Cell Based Assays in Drug
Discovery
Frank Stuhmeier, Ph.D., Senior Principal Scientist, Discovery Biology, Pfizer, Ltd. (UK)
Label-free technologies are currently not fully established in the mainstream early-stage drug discovery process, but can be used for cell line characterization, mode-of-action studies, SAR generation and primary screening. These technologies measure the overall response of a cell to an external stimulus and may become a valuable tool to improve the physiological correctness of assays. Over the last 18 months, Pfizer Sandwich has evaluated several plate-based label-free assay technologies. In my talk, I will outline the advantages and disadvantages of the different label free systems, and I will summarize some of the results. Furthermore, I will discuss the potential benefits of label free technologies, as well their integration into the drug discovery process.
11:55-12:20 Evaluation of Cellular Impedance Assays for Assessing Ligand-Specific GPCR Signaling in Drug Discovery
Matt Peters, Ph.D., Principal Scientist, Lead Generation, AstraZeneca Pharmaceuticals, Inc.
Subtle structural changes in GPCR agonists can change which down-stream signaling pathways are activated by a given receptor. The concept of ligand-specific signaling offers the potential to develop pathway-specific drugs with higher efficacy but fewer side effects. Despite the opportunity, currently functional selectivity is not widely exploited in drug discovery, perhaps due to the added difficulty of developing SAR for multiple signaling pathways. Cellular impedance assays are an emerging technology that may offer the ability to qualitatively distinguish Gs, Gi/o, and Gq signaling in a single assay. We have previously shown that the quality of the data generated by impedance assays is sufficiently robust to support drug discovery SAR studies investigating GPCR agonists, antagonists, and allosteric modulators. Here we extend this evaluation to specifically focus on the putative signal pathway-distinguishing ability of impedance response profiles. We find that CellKey impedance response profiles are highly dynamic and thereby encode valuable qualitative information on signaling. For example, GiPCR inverse agonists yield inverted profiles relative to GiPCR agonists and thus resemble the typical profile for GsPCR agonists. Similarly, receptors that activate multiple G-proteins simultaneously yielded novel intermediate response profiles. Finally, in tests on a panel of agonists reported to show assay/pathway-specific differential activity, we readily confirmed pathway-specificity even under traditionally challenging conditions such as human cells expressing native levels of receptor. These findings support broad use of impedance assays in SAR studies including ligand-specific signaling.
12:20-12:35 Sponsored Presentation (Opportunity Available.) Contact Katelin Fitzgerald, Manager, Business Development, at 781-972-5458 or kfitzgerald@healthtech.com.
12:35-1:00 Utility of Time-Resolved Binding Date for
Fragment-Based Screening and HTS Hit Validation Using SPR Biosensors
Anthony M. Giannetti, Ph.D., Research Scientist, Biochemical Pharmacology, Genentech, Inc.
SPR-based biosensors reveal the time-dependent interactions between molecules adding additional information about the interaction beyond just the potency. While traditionally used to characterize the binding kinetics, the time resolution of the assay can also be used to identify, characterize, and filter out pathological molecules such as promiscuous binders, which are a large source of the false positives from other screening modalities. Combining this information with the extreme sensitivity of the technology, we have leveraged the biosensor as a screening tool for fragment-based drug discovery (compounds with MW < 300 and KD as large as 5 mM) that can screen and verify hits from reasonably large fragment library (~5000 compounds) in a few weeks using < 1 mg of protein.
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FLIM-FRET for Live Cell Imaging
11:30-11:55 Dynamic Imaging of Protein-Protein Interactions in Living Cells: FLIM-FRET Microscopy
Ammasi Periasamy, Ph.D., Director, Keck Center for Cellular Imaging (KCCI); Professor, Biology and Biomedical Engineering, University of Virginia
Förster resonance energy transfer (FRET) methodology has been implemented in various sensitive fluorescence microscopy techniques to monitor protein-protein interactions in living or fixed specimens. On the other hand FLIM-FRET method provides direct evidence of interactions between proteins (Wallrabe and Periasamy, 2005). The FLIM system would reduce background interference and thus enhance measurement precision to yield more accurate understanding of protein associations in living cells. FLIM-FRET technology will significantly improve and expand existing capabilities for understanding the drug molecules interactions and for characterizing their binding properties as an ensemble and at the single molecule level.
11:55-12:20 Multidimensional Fluorescence Imaging
Paul French, Ph.D., Professor, Physics; Head, Photonics Group, Imperial College London
This talk will review the development and application of multidimensional fluorescence imaging (MDFI) technology, with an emphasis on fluorescence lifetime imaging (FLIM) optimised for high-speed imaging of live cells and applied to FRET studies of protein interactions. Applied to autofluorescence, MDFI can be used to provide label-free molecular contrast in biological tissue and is being developed for ex vivo and in vivo imaging applications. For cell biology and high-content analysis, we have developed an automated high-speed FLIM multiwell plate reader and a multiplexed FRET microscope capable of simultaneously imaging two different protein-protein interactions. For applications concerning embryos and small organisms, we have developed a FLIM optical projection tomography system.
12:20-12:45 Quantitative Molecular Sensing in Live Cells via FLIM
Mary-Ann Mycek, Ph.D., Associate Professor and Associate Chair, Dept. of Biomedical Engineering; Faculty Member, Applied Physics Program; Core Member, Comprehensive Cancer Center, University of Michigan
The ability to rapidly characterize molecular function in vivo would be a fundamental advance in biology and medicine. Fluorescence lifetime imaging microscopy (FLIM) is a technique that uses fluorophore lifetime rather than fluorescence intensity for image contrast. Compared to fluorescence intensity-based methods, fluorescence lifetime imaging requires less calibration and/or correction for fluorophore concentration, photobleaching, and other artifacts that affect intensity measurements. FLIM has been employed to probe the microenvironments of endogenous and exogenous fluorophores, including measurements of cellular metabolic co-factors, pH, dissolved gas concentration, and molecular interactions via FRET. Several applications of FLIM for quantitative, live cell imaging will be described, including studies of metabolic function and intracellular oxygenation, FRET studies of oncogene association, and microfluidic bioreactor characterization for continuous cell culture. Accurate image restoration approaches for FLIM will also be discussed.
12:45-1:10 Discrimination of Fluorescent Protein FRET Using Polarized Light
Mark A. Rizzo, Ph.D., Assistant Professor, Department of Physiology, University of Maryland School of Medicine
Fluorescence resonance energy transfer (FRET) is an exceptional tool for measuring protein-protein interactions in living cells. Adaptation of FRET assays to a high-content analysis platform is made possible by incorporating fluorescent proteins as labels. One limitation towards this goal has been the low contrast nature of conventional FRET assays. To circumvent this limitation, we have developed a novel approach for detecting fluorescent protein FRET by measuring the anisotropies of the fluorescent protein pairings. This approach yields high-contrast, unambiguous detection of FRET that is suitable for high content analysis.
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