Monday, January 14

7:30-2:00 Thermo Scientific User Group Meeting
2:30-7:00 GE Healthcare User Group Meeting
2:30-7:00 Molecular Devices User Group Meeting

5:00-7:00 Conference Pre-Registration

Tuesday, January 15

7:00-8:00 Conference Registration

8:00-8:15 Welcoming Remarks from Conference Director
Julia Boguslavsky, Cambridge Healthtech Institute

High-Content Screening
(joint session with co-located High-Content Analysis meeting)

8:15-8:40 Comparative Study of GPCR Internalization Assays
Sannah Zoffmann, Ph.D., Senior Scientist, Enabling Sciences, F. Hoffmann-La Roche Ltd.
Detection of GPCR internalization was one of the first applications established for image-based assays as a universal readout for receptor activation independent on G-protein coupling. Today there is increasing evidence that endocytosis might be only the default pathway following receptor activation but with exceptions. For various receptors agonists have been identified that do not induce endocytosis. This lack of endocytosis affects both, the overall signaling through G-protein dependent as well as independent pathways like MAP-kinase. Comparative, related in-vivo experiments suggest that such effects are responsible for desensitization leading to drug tolerance in chronic studies. As a consequence GPCR internalization has become an increasingly important tool in the drug discovery. Internalization assays complement the traditional binding and functional assays and facilitate differentiation and selection of early drug candidates. We have evaluated different approaches for the detection of GPCR internalization including chimeric receptor constructs, immunostaining, ligand tracking and finally the Transfluor assay. The presentation will summarize a head to head comparison of the different assays applied to the same GPCR.

8:40-9:05 Development and High-Throughput Screening of a High-Content GPCR Agonist Assay for a Complete Compound Library
Michael A. Nolan, Ph.D., Senior Research Scientist I, Inflammation, Wyeth Research
High-content analysis continues to grow in a wide number of both basic science and drug discovery research applications. This presentation describes the application of the Transfluor high-content GPCR internalization assay for hit identification in high-throughput screening of a large chemical library (~700,000 compounds). The assay was designed using the Cellomics ArrayScan V^TI then transferred to the PE/Evotec Opera confocal system to increase throughput. We will discuss assay development on both Cellomics and Opera platforms; validation runs with the LOPAC library; and hit identification, counter-screening and confirmation.

9:05-9:30 Biochemical and Cellular Pathway Screening Strategies: A Systematic Comparison
Jonathan Lee, Ph.D., Senior Research Advisor, Lead Generation and Optimization Biology, Eli Lilly & Co.
The pros and cons of using enzyme based biochemical assays versus cell-based signal pathway assays in lead generation/optimization are frequently discussed in the absence of direct, comparative studies. The Quantitative Biology group at Eli Lilly has utilized the p38 pathway to compare and contrast these screening modalities. 49K compounds were tested both in a coupled biochemical assay using p38-MK2 complex and a mechanistically related cell-based assay measuring the cytoplasmic/nuclear distribution of MK2. The cell-based assay produced 1300 primary actives. 1000 compounds were identified to be inhibitors of purified p38, IRAK4 or TAK1, known biochemical components of the interrogated signal transduction pathway. The cell-based signal pathway screen identified (1) three new p38 scaffolds, which have biochemical IC50 values < 300 nM and cellular IC50 values ranging from 100 nM to 4 uM, (2) thirty-five compounds that inhibit both the cell-based and biochemical MK2 assays, and (3) eleven compounds that inhibit the cell-based MK2 assay but did NOT significantly inhibit any of the kinase biochemical assays tested or translocation of an unrelated nuclear transcription factor. The strengths and weaknesses of biochemical and cellular pathway assays for lead generation and the implications of these results to Phenotypic Drug Discovery are discussed.

9:30-10:30 Coffee Break with Exhibit and Poster Viewing

10:30-10:45 New Technology Presentation
Advances in Cellular Systems Biology Cytotoxicity Profiling: 11-parameter Panel for HepG2 now Modified and Extended for Primary Rat Hepatocytes
Lawrence Vernetti, Ph.D., Director, Toxicology Program, Cellumen Inc.
Cellumen made significant advances in Cellular Systems Biology (CSB) profiling for predicting hepatotoxicity during the investigational safety phase. An 11-parameter, multiplex cytotoxicity panel used to investigate the HepG2 cell line was then compared and cross-validated with a similar CSB panel for primary rat hepatocytes in a study involving 110 compounds.

10:45-11:00 New Technology Presentation
(Sponsorship Available. Contact Carol Dinerstein at dinerstein@healthtech.com or 781-972-5471)

11:00-11:25 Development of a Homogeneous HIV-1 Entry Assay for Identification of gp41 Fusion Inhibitors
Marnix Van Loock, Ph.D., Scientist, Tibotec BVBA
Inhibitors of HIV-1 membrane fusion, the final step of the viral entry into a host cell, hold great promise to increase the effectiveness of antiviral therapy. Two "heptad-repeat" (HR1 and HR2) regions of the viral gp41 surface protein mediate fusion of host cell and viral membrane through obligatory formation of a six-helix bundle. To screen for compounds that inhibit HR1-HR2 interaction and subsequently viral entry, we developed a homogeneous competitive cell-based binding assay using persistently HIV-1 infected cells, which express envelope spikes containing a gp41 in its native confirmation. Upon exposure to the soluble cellular receptor of HIV-1 (sCD4), a fluorescent peptide fragment of HR2 (C34-FITC) specially stains the cell membrane in a dotted pattern. Indeed, sCD4 induces conformational changes in the spike and is a prerequisite to allow binding of C34-FITC to the HR1 region of gp41. This mimics the natural conformational changes that occur during the entry process. The assay was initially developed for FACS based read-out and was later transitioned onto a high-content screening system (OperaTM, Evotec Technologies). Development of the HCS assay using a suspension cell line in 384-well format was challenging in terms of image acquisition and homogeneous distribution of cells and reagents. We compare both fluorescent-based detection techniques for their assay quality parameters, the interference of fluorescent compounds and other artifacts, ability to extract cytotoxic information, costs and throughput.

11:25-11:50 Multiplex Analysis of a High-Content Cell-based Screen for Compounds Modulating VCAM1 Expression
Nathalie Aulner, Ph.D., Associate Research Scientist, Genome Center, Columbia University Medical Center
During inflammation, cytokine activation of the NFkB signaling pathway results in, among others, VCAM-1 (Vascular Cell Adhesion Molecule 1) cell surface expression. Adhesion and subsequent transmigration of circulating monocytes is carried out by VCAM-1 anchored to the cytoskeleton using its cytoplasmic domain. Failure to maintain an adequate cytoskeleton structure results in loss of monocyte adhesion. We have developed a high-content, cell-based assay that enables us to identify compounds that can affect eitherVCAM-1 expression or trafficking to cell surface. Concomitant staining of F-actin filament allows us to multiplex the output identifying compounds that perturb the cytoskeleton network and therefore potentially preventing VCAM-1 attachment and function even though it has been expressed properly. Moreover, via an automated high-throughput confocal microscope and suitable image analysis, it is also possible to detect any potential cytotoxic effect (cell count and effect on nuclear shape) identifying compounds triggering apoptosis for example. We have been able to identify several categories of compounds blocking several steps in VCAM-1 function, from its expression level to trafficking and perturbation of cytoskeleton anchor.

2:00-3:00 ThinkTank Roundtable Discussions
The concurrent roundtable discussions (open to all delegates) provide a small-circle forum for discussing key issues and meeting potential partners. The discussion facilitators will present an update the following morning. You must be a registered attendee to participate.

Screening with FLIM/FRET

3:00-3:25 Multidimensional Fluorescence Imaging
Paul French, Ph.D., Professor of Physics, Head of Photonics Group, Physics Department, Imperial College London
This talk will review the development and application of multidimensional fluorescence imaging (MDFI) technology including high speed fluorescence lifetime imaging (FLIM), to distinguish different molecular species, to sense the local fluorophore environment and to achieve label-free molecular imaging. Combining FLIM with tunable excitation sources and spectral and polarization-resolved imaging, we are able to resolve excitation and emission spectral as well as fluorescence decay profiles in a single data acquisition and to record 3-D polarization-resolved images to map protein binding and variations in viscosity. This technology is being applied to clinical imaging, for which we exploit autofluorescence to provide label-free contrast, drug discovery and molecular biology. Recent highlights include a high-throughput optically sectioning FLIM microscope system capable of imaging FLIM-FRET at up to 10 frames/second and the application of MDFI to microfluidic devices.

3:25-3:50 High-Content Screening of Intracellular Processes by High-Speed FLIM and FRET
Tony J. Collins, Ph.D., Assistant Professor, McMaster Biophotonics Facility, Department of Biochemistry and Biomedical Sciences, McMaster University
The most pressing needs in high-content screening include ways of rapidly and accurately monitoring biological processes at the cellular and subcellular level, particularly in live cells. One of the most promising approaches in monitoring specific enzyme activity, intracellular ion concentrations, and protein-protein interactions is FRET (fluorescence resonance energy transfer). One problem faced by many researchers is that measuring FRET in an intensity-based image requires complex cross-talk corrections, extensive spectral characterization, or extended periods of photobleaching the acceptor fluorophore. However, quantifying FRET using fluorescence lifetime avoids many of the pitfalls and long acquisition times associated with conventional intensity-based approaches. Novel fluorescence lifetime imaging techniques that can be used to quantify enzyme activity by intermolecular FRET and the interaction of two protein partners by intramolecular FRET, with sufficient throughput for high-content screening, will be described.

3:50-4:15 Patch Fluorometry: Focus Fluorescence Light on Drug Target Proteins in Plasma Membrane
Jie Zheng, Ph.D., Assistant Professor, Physiology and Membrane Biology, University of California
HTS has traditionally relied heavily on electrophysiology, which is limited to current-generating ion channels and drug candidate molecules that bind from the extracellular side. These limitations can be easily overcome with optical readouts. I will highlight two complimentary fluorescence techniques that we developed in recent years. Patch Fluorometry is a membrane patch-based assay allowing accurate, sensitive, and robust monitoring of the activity of membrane proteins (channels and receptors). Spectra FRET is a cell-based assay that allows plasma membrane-specific measurements. Potential applications of these latest developments in drug discovery will be discussed.

4:15-5:10 Refreshment Break with Exhibit and Poster Viewing

Advances in Fluorescent Probes and Biosensors

5:10-5:35 New Windows on Living Cells: Biosensor Designs
Klaus Hahn, Ph.D., Ronald Thurman Distinguished Professor of Pharmacology, Department of Pharmacology, UNC
This talk will cover new tools to study protein conformational changes in living cells, and demonstrate their utility by revealing the dynamics of Rho family GTPase signaling during motility and transendothelial migration. The talk will cover methods to study endogenous protein activity, simultaneous imaging of coordinated signaling activities, and biosensors for previously inaccessible targets made via phage display screening of biosensor libraries.

5:35-6:00 Fluorescent Probes for Drug Discovery
Alan Waggoner, Ph.D., Professor, Biological Sciences; Director, Molecular Biosensor and Imaging Center, Carnegie Mellon University
The tools of fluorescence detection include fluorescent labels, physiological indicator probes and, more recently, fluorescent protein biosensors. Fluorescent proteins have become a widely used technology in basic research and drug discovery assays. Fluorescent proteins can be genetically encoded or incorporated into living cells from the medium. Flow cytometers, imaging microscopes and high density plate readers are used to read out signals. Regulation of cell structure and function depends on the concerted activity of thousands of proteins within living cells. A big challenge remains for developing and using new fluorescent protein biosensor to sort out the detailed molecular interactions of these proteins as they go about their business. Protein-protein interactions, protein modification, conformational change, activity change, locality change, expression and degradation are all targets for biosensor development. This presentation will cover a number of new directions in fluorescent biosensor development including those in the speakers laboratory at Carnegie Mellon University.

6:00-7:00 Reception with Exhibit and Poster Viewing

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