8:30-8:55 Recap of Roundtable Discussions

8:55-9:00 Chairperson's Opening Remarks
Dr. Judy Masucci, Dir Product Marketing, Product Marketing, Cellomics, Inc.

9:00-9:30 Application of RNAi, High-throughput Transfections and High Content Biology
to the Target Validation Process
Dr. Judi Wardwell-Swanson, Sr. Reasearch Investigator II, Applied Genomics Dept., Bristol-Myers Squibb
Until recently target validation could be characterized as a slow process involving the prosecution of single targets through a myriad of biochemical and cell based assays. Recent advances in the area of gene silencing and cell-based screening have made possible a higher throughput and more contextually relevant approach to target validation. A target validation platform that combines the efficient and specific silencing of individual mammalian genes by siRNA with a wide variety of multiplexed high content screening endpoints could soon represent the perfect complement to microarray-based expression profiling

9:30-10:00 siRNA Gene Silencing and High-Content Analysis for Target Validation
Dr. Sergey Ilyin, Bioinformatics Group Leader, Johnson & Johnson Pharmaceutical Research & Development
Novel paradigm: Functional Informatics, a convergence and integration of Bioinformatics and Automation for target functional identification and validation. In this new informational model, we took advantage of existing HTS equipment and combined it with rationally designed libraries of small interfering RNA (siRNA) molecules to perform automated high-content cell-based screening.

10:00-10:30 Characterization of the Human Kinome through High-Content siRNA Screens
Dr. Mark R. Lackner, Research Scientist, Signal Transduction Research, Exelixis, Inc.
We are engaged in creating a database of knockout phenotypes (by RNAi) for each human kinase in multiple cell based assays, including Erk and c-Jun phosphorylation, apoptosis, and proliferation. These efforts will not only serve in target identification, but will also serve to guide the choice of assays and cell types for validation studies on individual kinases. Methods for medium throughput screening of siRNA libraries in multiplex single cell assays, as well as issues around correlation of protein knockdown with cellular phenotype, will also be discussed.

10:30-11:10 Coffee Break with Poster and Exhibit Viewing

11:10-11:15 Chairperson's Opening Remarks
Dr. Jonathan Lee, Director, Discovery Technologies, Eli Lilly and Co.

11:15-11:45 High Content and High Information Cell-Based Assays
Dr. Jonathan Lee
In the Pharma Research environment, a successful new technology must provide information about novel activities or novel cell systems that other assay methodologies cannot directly or indirectly measure. In Discovery Technologies, we have utilized cell sorting, multiplexed analyte detection, proteomics, and fluorescence imaging to develop novel high-content and high-information cell based assays applicable to heterogeneous cell populations, biomarker discovery, toxicology, and mechanism of drug action. Select examples will be discussed.

11:45-12:15 Bridging the Gap Between Classical In Vitro Assays and High Resolution Live Cell-Based Assays: The Multiplexed Cell-Based Assays
Dr. Claudine Grepin, Head of Assay Development, Lead Discovery Technology, Aventis
Between the conventional in vitro biochemical assays and the high level of information provided by live cell-based assays analyzed at the subcellular level, lies the cellular multiplexed assay which complements the battery of formats available for both primary and secondary screening. Having implemented High Content Screening using Acumen® for both primary and secondary assays, Aventis is now moving to multiplexed cell-based screening technologies because they combine the functional relevance of cell-based assay, the throughput and robustness of in vitro format while keeping the high content of information. The data obtained with two multiplex systems evaluated for the measurement of multiple phosphoproteins or multiple mRNAs present in cells after compounds treatment in standard 96/384 wells will be presented. The potential impact of the integration of these technologies in HTS will be discussed in light of the value that the HCS have added to the Aventis' drug discovery process during the last years.

 

12:15-12:45 High-Content Cellular Assays Chairperson: Dr. Jonathan Lee, Director, Discovery Technologies, Eli Lilly and Co. Panelists: Dr. Claudine Grepin, Head of Assay Development, Lead Discovery Technology, Aventis Ms. Ann F. Hoffman, Senior Principal Scientist, Cell-Based HTS, Hoffmann-La Roche Inc. Dr. Lekha Patel, Senior Scientist, Drug Discovery, Exploratory Technology, Johnson and Johnson Dr. Jeffrey Haskins, Vice President of Assay Development, Cellomics, Inc. Discussion topics: What does "High-Content" exactly mean? In addition to high-resolution imaging, does "High Content" include analyte multiplexing and multi-para metric approaches? Are image-based high-content assays currently offering unique information? What are the true advantages of "High-Content" cell based assays? Where do "High-Content" assays fit into the drug discovery pipeline? What are the potential applications of "High-Content" cellular assays? What are the infrastructure "hidden costs" in image-based high-content assays? Are there benefits to kinetic vs. end-point assays?

12:45-2:15 Luncheon Technology Workshop	Sponsored By:
Quantitative Microscopy; A Systems Solution for High Content Screening Presented by David Hanzel, Ph.D. and Dietrich Ruehlmann Ph.D.

Informatics for HCS: Image Analysis and Data Management

2:15-2:20 Chairperson's Opening Remarks
Dr. J. Paul Robinson, Professor of Immunopharmacology and Biomedical Engineering, Purdue University

2:20-2:50 Technology Integration for Analysis of High Throughput Cellular Data: The Cytomics Approach
Dr. J. Paul Robinson
This presentation will discuss current ideas for analysis of live cell data incorporating multivariate approaches. It will outline the major problems faced by present generation technologies and provide insight into future advances. Key to the success of future technologies will be an understanding of informatics and high-speed data processing including advanced image analysis.

2:50-3:20 Informatics for High Content Screening
Mr. Mike Esterman, Sr. Information Consultant, Discovery IT, Eli Lilly and Co.
High Content Screening based on imaging of cellular events has become an important tool for drug discovery because of the speed and the ability to multiplex. In this talk I will discuss the informatics issues introduced by the extremely high volume of data produced by HCS technology from the perspective of a pharmaceutical company. This talk will be an overview of image analysis, storage and retrieval of data, long term storage, and 21 CFR Part 11 compliance.

3:20-3:50 Informatics for High-Content Cellular Analysis Chairperson: Dr. J. Paul Robinson, Professor of Immunopharmacology and Biomedical Engineering, Purdue University Panelists: Mr. Mike Esterman, Sr. Information Consultant, Discovery IT, Eli Lilly and Co. Dr. Jay Gill, Group Leader, Informatics, Bristol-Myers Squibb Dr. Mark Collins, Senior Director of HCS Informatics, Cellomics, Inc Discussion Topics: Data: How to get terabytes of data from instrument to secure location? How to identify storage size need and bandwidth? How to identify level of security? Is the data in a proprietary format? Implications? 21 CFR Part 11 compliance Image Processing: Do you secure the primary images? Secondary/daughter and subsequent images? How do you store and identify the processing algorithms? What methodology is used to track process used for analysis? Standards and calibration: Are the data collected against a national standard? Are there adequate internal standards? Are the parameters standard (can be 10-80 parameters)? Are there guidelines for secondary analysis of these parameters? Is there good reproducibility within/between systems? Image processing programs: Are you locked into a proprietary package? Are the analysis components within a system locked into the instrument vendor ? What is the required image resolution for HTS? Should data be 8-, 12-, 16-bit image? What magnification should be used (10, 40x)? Is large field more useful than high resolution?

3:50-4:15 Refreshment Break with Poster and Exhibit Viewing

 

4:15-6:15 New Technology Showcase Chairperson: Dr. Susan Catalano, President, Drug Discovery Imaging 4:15 Modulation of Protein Translocation as an Alternative to Inhibition of Catalytic Activity: Selective Hits from Redistribution Technology Dr. Len Pagliaro, VP Discovery Projects, BioImage A/S We have recently screened chemical libraries against Redistribution targets in the PI-3-kinase and p38-MAP kinase pathways, and we have identified families of selective compounds with therapeutic potential. Redistribution is a discovery approach developed for identifying compounds that specifically modulate the function of individual intracellular signaling proteins without affecting their catalytic activity. We believe that our newly identified leads demonstrate that Redistribution is a viable strategy for early discovery of novel, selective drug candidates that act at difficult pathway targets. 4:30 High-Throughput Cell Imaging and Manipulation Using the LEAP™ Platform Dr. Fred Koller, Vice President of R&D and COO, Cyntellect, Inc. The novel, high-throughput Laser-Enabled Analysis and Processing (LEAP™) platform couples real-time laser-based cell manipulation capabilities with high-content imaging, thereby enabling a new generation of interactive active cell-based assays. Active assays employ in situ laser-mediated optoinjection (for transfection), cell purification (to eliminate untransfected cells), and subsequent high-content imaging, allowing development and implementation of an end-to-end assay within one well to circumvent laborious and time consuming cell manipulations required in most currently practiced procedures. 4:45 Using Cell-Based Reporter Assays to Decipher Signaling Complexity Dr. Mehran Khodadoust, President and Chief Scientic Officer, Bionaut Pharmaceuticals By using positive selection (in the presence of an agent of interest) followed by negative selection (to eliminate lines where the reporter gene is regulated by housekeeping promoters), Bionaut generates cell lines with reporter genes specifically regulated by selected signal transduction pathways. The Sentinel Pathway Reporter System permits dissection of precise causal relationships throughout a regulatory pathway without prior knowledge of the pathway’s complexity. This allows early and rapid screening for efficacy, as well as pathway redundancy, specificity and adverse side-effects — the latter issues responsible for the substantial portion of drug failures during downstream clinical trials. 5:00 High-Throughput Microscopy for Intracellular Event Visualization in Drug Screening Dr. Jeffrey Price, Founder and Chief Scientist, Q3DM Inc., and Associate Research Scientist, Dept. of Bioengineering and Whitaker Institute of Biomedical Engineering, University of California, San Diego High-throughput microscopy (HTM) techniques are proving valuable in drug discovery for evaluating not just the response of a cell population to a compound, but also subcellular pathway stimulus responses on each cell. With the ability to image entire populations at higher resolutions (20 ­ 60x up to 0.95 NA), observation of intracellular events, like subcellular compartment translocation, aggregate formation, and multiple fluorescent reporter co-localization, and transcription is enabled. Acquisition of high-content data (from images) on each cell over large populations has been combined with efficient data mining tools for relating specific cell subpopulation image galleries to ranges of cellular measurements; this visual virtual cell sorting increases confidence in candidate drug molecule selection and will reduce late-stage attrition by creating not only a yes/no or dose response but visualization of the target localization and pathways within the signaling networks. These advanced capabilities will be demonstrated with several applications and results from validation studies for subcellular screens including: NFκB nuclear translocation and GPCR activity assays, imaged and analyzed on the Q3DM Eidaq™ 100 HTM system. 5:15 Pharmacological Profiling of Drugs in Living Cells Dr. John K. Westwick, Vice President of Drug Discovery, Odyssey Thera, Inc. By detecting how a molecule affects specific steps within a biochemical pathway, the mechanism of action of a novel compound can be determined, allowing lead optimization to proceed. Odyssey Thera has used its fluorescent high-content assays to profile 60 known drugs and a diverse set of siRNAs against a spectrum of signaling pathways in living cells. This strategy, which we call KUDOS (Known/Unknown Drug Optimization Strategy)is designed to enable the systematic, biology-based screening for unintended or off-pathway effects of lead compounds in living cells. 5:30 Automated HCS Image Analysis Technology Dr. John Elling, President and CEO, Cytoprint, Inc. Cytoprint has developed High Content Screening image classification technology. The method generates a standard, high dimensional image descriptor that is sensitive to changes in cellular images that results from compound treatments. Statistical analysis of the descriptors allows identification of the image changes that are signals of biological effects. 5:45 Reverse Transfection Cell Microarrays in Drug Discovery and Development Dr. Craig Thompson, Group Leader, Cell Biology, Akceli, Inc. Cell microarrays are created by reverse transfection of nucleic acids that have been printed in discrete array patterns on the bottom of 96-well plates. The result is an array of distinct cell clusters, each of which produces a protein encoded by an expression vector spotted at a given feature or is deficient for a specific protein as a result of siRNA-mediated mRNA degradation. Coupling these arrays with cellular phenotypic readouts (e.g. mitotic arrest, apoptosis, viability) and protein class specific readouts (e.g. tyrosine kinases, GPCRs) has enabled multiplexed target validation and screening across a variety of therapeutic areas and target classes. 6:00 Single-Well Determination of Potency and Selectivity Using the CellCard Platform Dr. Oren Beske, Head of Cell Biology, Vitra Bioscience The drive for more biologically relevant information with predictive power has resulted in the advent of new cell-based assays and platforms that enable scientist to gather increasing amounts of information from each experiment. The CellCard platform combines multi-parameter cellular analysis and selectivity screening within a single microtiter well. This provides the ability to simultaneously profile a compounds activity across multiple parameters as well as across multiple cell lines within the confines of a microtiter well ensuring identical assay conditions 6:15 Cellular Analysis Platform with Dynamic Fluorimetry for Drug Discovery Dr. Evan F. Cromwell, President, Blueshift Biotechnologies, Inc. The drug discovery industry is in need of improved screening technologies that provide knowledge and foresight into success of new drug candidates. One of the more recent developments in optical imaging for complex biological systems is the use of fluorescence lifetime imaging microscopy (FLIM) and fluorescence polarization (FP). FLIM and FP provide powerful tools for assays that use detection of the presence, quantity, and location of labeled molecules and their binding state. Blueshift Biotechnologies’ Dynamic Fluorimetry platform simplifies these measurements by exploiting recent advances in laser sources, semiconductor inspection technology, fluorescent probes, and high content screening. This platform will enable new approaches for cellular analysis.