DISCOVERY OF TARGETS AND LEADS

8:30 Chairperson’s Remarks
Dr. Alan S. Verkman, Professor of Medicine and Physiology, University of California San Francisco

8:35 Live Cell-Based Assays for Compound Screening
Dr. James Inglese, Associate Investigator, Director of Biomolecular Screening & Profiling, Genome Technology Branch, National Institute of Health, National Human Genome Research Institute

9:05 Use of Fluorescent Proteins for Early Lead Identification Strategies
Dr. Bonnie J. Howell, Senior Research Biochemist, Cancer Research, Merck & Co. 
Automated fluorescence microscopy, image analysis, and screening technologies are some of the most powerful tools enabling cell biologists to examine complex signaling pathways and cellular response. Through the use of multiple fluorescent tools, we are able to track cellular activity, measure phenotypic response, and provide contextual information about cellular pathways and off-target effects. We are using these capabilities to achieve high-content screening of cellular and molecular fluorescent endpoints in vitro following drug treatment. Case studies will be presented describing the use of fluorescent proteins for monitoring cell cycle stage and protein translocation, and these methods will be compared to other fluorescent labeling techniques.

9:35 Imaging Fluorescent Proteins in Living Fungal Cells
Dr. Patrick Hickey, Biological Sciences, Edinburgh University, and LUX Biotechnology Ltd
Fluorescent proteins in combination with vital fluorescent dyes are
powerful tools enabling researchers to non-invasively interrogate living cells. An overview of confocal fluorescence imaging technology and the advantages of utilizing fungal cells for high content screening (e.g. toxicity, drug testing and antifungal discovery) will be covered. The presentation will include a selection of images, 3-D reconstructions and time-lapse movies (e.g. vesicle trafficking, organelle dynamics and hyphal growth) whichillustrate the unique properties of filamentous fungi.

10:05 Coffee Break, Poster & Exhibit Viewing

10:45 CFTR Drug Discovery Using Halide-Sensing GFPs
Dr. Alan S. Verkman
Cystic fibrosis (CF) is a relatively common hereditary disease causing chronic lung infection leading to early death. CF is caused by defects in the CF transmembrane conductance regulator (CFTR) chloride channel. The most common CFTR mutation causing CF is the F508 mutation, which has defects in cellular processing with ER retention and in channel gating. Cell-based fluorescence assays of halide flux using iodide/chloride-sensing GFPs were developed for identification of F508-CFTR potentiators (which correct defective gating) and correctors (which correct defective processing), as well as CFTR inhibitors (for therapy of enterotoxin-mediated secretory diarrheas). Potent activating and inhibiting compounds were discovered and characterized.

11:15 New Fluorescent Protein Transfection Models for in Vitro and in Vivo Studies of Human Hepatoblastoma and Pediatric Rhabdomyosarcoma
Dr. Steven W. Warmann, Pediatric Surgery, University Children’s Hospital 
Creation of cell line models for human hepatoblastoma (HB) and pediatric rhabdomyosarcoma (RMS) using GFP and DS2Red. Xenotransplantation of cell clones into nude mice NMRI (nu/nu) followed by fluorescent imaging assessments. Analysis of the biological behavior of tumor cells in vitro and in vivo. Assessment of treatment approaches of transfected tumors in vitro and in vivo.

11:45 Image-Based Screens for Cell Cycle and Cancer Targets
Dr. Daniel R. Rines, Institute Fellow, Lead Discovery, Genomics Institute of Novartis 
Using a RNA library of 49,000 double-stranded (ds)RNAs targeting approximately 24,000 genes, we performed a loss-of-function screen for essential mitotic chromosome segregation genes. We identified novel genes whose inactivation caused mitotic arrest. Multi-parametric analysis of image-based data derived from a high-content screen including phospho-histone H3 levels (p-His), cellular proliferation and nuclear morphology allowed us to isolate both checkpoint dependent and independent segregation genes.

12:15 Lunch on your own or Technology Workshop
(Sponsorship Available)

COMPARISON OF FLUORESCENCE AND LUMINESCENCE

1:30 Chairperson’s Remarks
Dr. Keith Wood, Research Fellow, Promega Corp.

1:35 Quantitative Comparison of the Detection Sensitivity of Bioluminescent and Fluorescent Reporters in Animal Models
Dr. Tamara Troy, Senior Scientist, Xenogen Corporation
Both bioluminescent and fluorescent reporters can be detected and monitored in vivo and each has different strengths and weaknesses, depending on the specific applications. Given the widespread use of light emitting probes, there is a need for more quantitative comparison of the tradeoffs in terms of sensitivity, spatial localizations and ease of use. In the work presented here, an overview of a dual-reporter imaging system is presented and in vivo comparisons are shown.

2:35 A Cell-Based cGMP Assay Useful for Ultra-High-Throughput Screening and Identification of Modulators of the Nitric Oxide/ cGMP Pathway
Dr. Frank Wunder, Institute of Cardiovascular Research, Bayer HealthCare AG
We have established a rapid, homogeneous, cell-based, and highly sensitive assay for guanosine 3’-5’-cyclic monophosphate (cGMP) that is suitable for fully automated ultra-high-throughput screening. In this assay system, cGMP production is monitored in living cells via Ca2+ influx through the olfactory cyclic nucleotide-gated cation channel CNGA2, acting as the intracellular cGMP sensor. A stably transfected Chinese hamster ovary (CHO) cell line was generated recombinantly expressing soluble guanylate cyclase, CNGA2, and aequorin as a luminescence indicator for the intracellular calcium concentration. This cell line was used to screen more than 900,000 compounds in an automated ultra-high-throughput screening assay using 1536-well microtiter plates. In this way, we have been able to identify BAY 58-2667, a member of a new class of amino dicarboxylic acids that directly activate soluble guanylate cyclase. The assay system allows the real-time cGMP detection within living cells and makes it possible to screen for activators and inhibitors of enzymes involved in the nitric oxide/cGMP pathway.

3:05 Refreshment Break, Poster & Exhibit Viewing

3:35 Generation of Light Producing Assays for Enzymes Involved in Drug Metabolism
Dr. John Shultz, Group Manager, R&D, Promega Corp.
Fluorescent detection techniques are commonly used for determining the localization of proteins within cells. Light producing reactions, on the other hand, are used to monitor various cellular events, such as induction or repression of protein expression. Until very recently it was impractical to use a Luciferase mediated light signal for the measurement of various enzyme activities since the substrates needed for light production were either used exclusively by Luciferase (such as luciferin) or were ubiquitous to the cell (such as ATP). This presentation will describe various strategies that have been used for producing assays for various enzymes involved in drug metabolism that are coupled to the generation of a light signal produced by Luciferase. In the future, such coupled assays may allow imaging in animals. 

	4:05 Selected Poster Presentation
	4:20 Selected Poster Presentation

4:35 Close of Fluorescent Proteins Conference