CONCURRENT SESSIONS

ASSAY DESIGN FOR HCA

8:30 Chairperson’s Opening Remarks

8:30-9:00 A Comparison of Different High-Content Methodologies as Applied to Lead Discovery and Target Validation
Mrs. Judy Dziuba, Senior Scientist, Lead Discovery, Bayer Healthcare AG
The Bayer Lead Discovery group in West Haven has many and varied functions including assay development, biochemical and cell based high-throughput screening, hit confirmation and the management of a million compound library. Introduction of High-Content Image Methodologies has complemented and enhanced cell-based HTS, lead characterization, and target validation efforts. We have adopted the TTP Labtech Acumen Explorer and the BD/Atto Pathway for these purposes and their utilities will be discussed in an application-specific context. The utility of laser scanning as applied to high-throughput screening will be examined and it will be compared to a more traditional cell ELISA and to high-content image based analysis.

9:00-9:30 Assay Development for the Targeting of Distinct Cellular Phenotypes
Dr. Oliver Poeschke, Scientist, Central Assay Development & Screening, Merck KGaA
The cellular phenotype can be analyzed on the levels of cellular growth morphology, single cell texture changes and sub cellular events. Quantification of small-molecule driven phenotypic modulation highly demands the development of robust screening assays. We will present our experiences in the development of assays for different target classes (GPCRs and kinases) as well as cellular mechanisms (e.g. cell cycle analysis). We will show that the use of antibodies, fluorescent dyes or reporter proteins in combination with the Acumen HT reader as technology platform allows the supply of robust high-content screening assays.

9:30-10:00 An Integrated Robotic Platform for High-Content Screening: Analysis of GPCR Internalization and Cellular Signaling
Dr. Ralf Heilker, Senior Scientist, Integrated Lead Discovery, Boehringer Ingelheim Pharma GmbH & Co. KG
We have integrated the IN Cell Analyzer 3000TM into a dedicated, fully automated robotic platform for high-content screening (HCS). The application of the robotic system is illustrated by the example of automated dose response testing for AKT1-signaling. In applying HCS to GPCR functional analysis, we observed a correlation between GPCR internalization kinetics and GPCR ligand efficacy. Various reference agonists for the ß2 adrenergic receptor were compared on this basis.

NEURONAL SCREENING

8:30 Chairperson’s Opening Remarks

8:30-9:00 Applying the Power of HCA to Neuronal Screening
Dr. John Dunlop, Director, Discovery Neuroscience, Wyeth Research
The introduction of high-content screening applications for quantification of neurite outgrowth has provided a powerful tool toward the discovery of novel agents with neuroprotective and regenerative properties. In addition to target-based approaches this platform allows for non-biased phenotypic screening in a format compatible with small and medium sized compound libraries. New developments in target-based and phenotypic based screening will be presented.

9:00-9:30 Screening Chemical Libraries to Identify Regulators of CNS Axon Growth and Branching
Dr. Lynn Usher, Research Associate, The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine
Following spinal cord injury, CNS neurons encounter an environment hostile to axonal regeneration, resulting from a relative lack of growth-promoting cues and an abundance of growth-inhibitory cues. We are taking two complementary approaches to understanding interactions of neurons with this environment and changing the balance in favor of regeneration. In one approach, we are screening a combinatorial library of organic compounds to identify those that promote neurite outgrowth of primary central nervous system neurons, on an inhibitory substrate (myelin) that mimics the injured spinal cord. Four compounds have been found with appropriate activities in this assay. In a second approach, approximately 500 compounds with known bioactivities are being screened on these primary neurons for differential effects on axon growth vs. branching, and on growth/branching stimulated by distinct cell adhesion molecules (CAMs). Initial results in this screen have identified novel pathways that underlie neurite growth stimulated by the different CAMs.

9:30-10:00 Technologically Integrated Assay Approaches to Complex Signaling Events 
Mr. Oscar Trask, Associate in Research, Center for Drug Discovery, Duke University Medical
High-Information-Content Kinetic Measurement of N Advances in high-content imaging technology has facilitated the development of multiple endpoint and kinetic screening approaches to intracellular signaling in neuronal cultures. More recently, combining these sophisticated imaging assays with multi-electrode array electrophysiological recording should begin to facilitate further improvements in our understanding of the regulation of ionic signaling and synaptic physiology euronal excitability

10:00-10:30 Coffee Break

10:30-11:00 Phenotypic Assays for Oncology Drug Discovery
Dr. Bonnie Howell, Senior Research Biochemist, Cancer Research, Merck & Co., Inc.
Automated fluorescence microscopy, sophisticated image analysis software, and novel screening technologies are some of the most powerful tools enabling cell biologists to investigate complex signaling pathways rapidly in individual cells. Using these tools, we are able to track cellular activity, measure phenotypic change, and provide contextual information about cellular pathways not discernable through traditional single-parameter experiments. Through the use of multiple probes, we are able to quantify changes in cell proliferation, cell cycle stage, apoptosis, and morphology, as well as measure off-target effects within a single assay. Using this approach in combination with data visualization tools, we are generating heat maps of cellular response and increasing our understanding of the molecular and cellular changes that occur upon chemical perturbation, and the heterogeneity in cellular response within and between cell populations.

11:00-11:30 Developing and Validating a High-Content Screen for Intracellular Cholesterol Distribution
Dr. Fred Maxfield, Professor and Chairman, Department of Biochemistry, Weill Medical College of Cornell University
The intracellular transport of cholesterol is of great importance in normal physiology and in various diseases. We developed a screening procedure to look for compounds that would correct the gross cholesterol storage defect in CHO models of Niemann-Pick C (NPC) disease, a lysosomal storage disorder in which large amounts of cholesterol are stored intracellularly. We developed an assay based on staining cells with filipin, a fluorescent detergent that binds to unesterified cholesterol. When tested on NPC vs. control CHO cell lines, an assay that analyzed sub cellular distribution as well as brightness provided very good discrimination (Z’ > 0.6). An automated screening protocol for 384 well plates was developed and used to screen a diverse library of 15,000 compounds. We found 14 compounds that reproducibly reduced filipin staining at 10 ?M, including 3 that were effective at 120 nM.

11:30-12:00 High-Content Analysis for DNA Content
Mr. Aidas Kriauciunas, Research Scientist, Integrative Biology, Eli Lilly and Company
The distribution of cells in the G1, S, and G2/M phases of the cell cycle can be determined by measuring nuclear DNA content. Fluorescent dyes like propidium iodide bind to DNA in a stoichiometric manner and emit a fluorescence signal that is proportional to the amount of DNA in the nucleus. Flow cytometry has been the gold standard for measuring DNA content of cells for over 30 years. However, it’s not ideally suited for use with adherent cells and affords limited throughput. Therefore, a 96-well method for measuring DNA Content was developed and validated using the Acumen Explorer. Using this approach, we were able to obtain detailed concentration response curves of known inhibitors that were in agreement with flow cytometric results and provided an approximate 10-fold increase in throughput as compared to the traditional flow technique.

12:00-12:30 Finding Content in High-Content Data : Two Case Studies for Target-Specific and Phenotypic HCS Assays
Dr. Andreas Vogt, Research Assistant Professor, Pharmacology, University of Pittsburgh
Image-based single-cell multiparametric analysis has evolved to a point where high-throughput, multiplexed analysis of thousands of individual cells is technically feasible. However, extracting meaningful data from high-content single-cell analyses can represent a challenge, in particular when large numbers of parameters per cell are measured. Here we will illustrate how thoughtful assay design and innovative data analysis have resulted in the implementation of a target-specific screening assay for mitogen-activated protein kinase phosphatase-1 inhibition and the discovery of a putative cellular target for the natural product, Disorazole C1.

10:30-11:00 High-Content Secondary Screening of Apoptosis in Neuronal Cell Lines
Dr. Dorothea Haasen, Research Scientist, Integrated Lead Discovery, Boehringer Ingelheim Pharma GmbH&Co. KG
With respect to neuronal cell death, we have validated a number of high-content readouts in multiplex assays, analyzing markers of programmed cell death with the IN Cell Analyzer 3000TM as a high-content screening instrument. Standardization of these assays will allow higher-throughput on a fully automated robotic platform. Kinetics, concentration dependency of apoptosis induction as well as classification of different stimulators will be discussed.

11:00-11:30 High-Content Analysis of Human Embryonic Stem Cell Growth and Pluripotency
Dr. Paul Sammak, Associate Professor, Pittsburgh Development Center at the Magee Women’s Research Institute, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh 
Human embryonic stem cells (hESC) are a promising source for well characterized tissue-specific cells for compound screening and toxicology. However, standardization of cell culture practices, quality assurance standards and criteria for hESC are still evolving. We have used automated high-content analysis of single cells and whole colonies to develop criteria for pluripotency, growth and death rates that can be used to optimize environmental conditions during hESC maintenance and neuronal differentiation. Novel algorithmic approaches provide non-invasive methods for evaluating hESC colony pluripotency and heterogeneity and provide a basis for reproducible colony propagation.

11:30-12:00 Development of an In Vitro Model for Nerve Injury on the Discovery-1 Platform
Dr. Mark Schurdak, Group Leader, Biological Screening, Abbott Laboratories
CNS injury results in the formation of glial scars that create an environment which is non-permissive for axon regeneration.   Growth inhibitory substances present in the glial scar have been shown to function through activation of the Rho/Rock pathway, which results in growth cone collapse, and among the most potent growth inhibitors in the glial scar are the chondroitin sulfate proteoglycans (CSPGs).   Using NGF differentiated PC12 cells, we have established an in vitro nerve injury model wherein the Rho/ROCK pathway is activated by CSPG, and the effects of test compounds on reversing growth inhibition are quantified using the Discovery-1 system.  This model is being employed to screen kinase inhibitors identified in an HTS campaign with the goal of using a more physiologically relevant assay to identify potent compounds with the appropriate cell activity to move into animal models. 

12:00-12:30 Automated Fluorescent Imaging in Live Brain Tissue Explants: Challenges for Ultra-High-Content Screening (HCS) in Drug Discovery for Neurodegenerative Diseases
To Be Announced 
We have developed a novel approach to quantify neuronal health and viability in live brain tissue explant models for neurodegenerative diseases including Huntington’s disease and Alzheimer’s disease. These models for neurological disease, based on cultured rodent brain slices into which human disease genes have been introduced via particle-mediated gene transfer (“biolistics”), are being used for high-content screening for therapeutic small molecule drug candidates and drug targets. In this talk I will discuss the challenges and approaches used in optimizing the development and analysis of live explant tissue imaging using stereomicroscopy and the Cellomics Arrayscan VTI HCS Reader. The results of this study demonstrate the capabilities of the Zeiss AxioVert 200M inverted fluorescent microscope in imaging individual neurons and specific neuronal features in 250 micron-thick live brain slices using “macro”-like optics, and the ability to use such measurements in the design and operation of small molecule compound screening in live brain tissues. These results indicate that commercial development and innovation will be effectual in supporting the exploration of non-conventional, tissue-based screening platforms. Furthermore, these findings should help promote future assay development in live ex-vivo tissue and whole model organisms using High-Content screening and High-Content analysis approaches. Supported by the Cure Huntington’s Disease Initiative and the National Institute of Neurological Disorders and Stroke (NS048181).