8:40-9:05 Integrating High-Content Screening and Ligand-Target Prediction to Identify Mechanism of Action
Yan Feng, Ph.D., Lab Head, Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research
High-content screening (HCS) is transforming drug discovery by enabling simultaneous measurement of multiple features of cellular phenotype that are relevant to therapeutic and toxic activities of compounds. HCS studies typically generate immense datasets of image-based phenotypic information, and how best to mine relevant phenotypic data is an unsolved challenge. Here, we introduce factor analysis as a data-driven tool for defining cell phenotypes, and profiling compound activities. This method allows a large data reduction while retaining relevant information, and the data-derived factors used to quantify phenotype have discernable biological meaning. We use factor analysis of cells stained with fluorescent markers of cell cycle state to profile a compound library, and cluster the hits into seven phenotypic categories. We then compare phenotypic profiles, chemical similarity and predicted protein binding activity of active compounds. By integrating these different descriptors of measured and potential biological activity, we can effectively draw mechanism of action inferences.

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.

8:40-9:05 Genome-Wide Multi-Parametric Endocytosis Screen Reveals Pitfalls of siRNA Design and Technology
Eberhard Krausz, Ph.D., Head, HT-Technology Development Studio (TDS), Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG)
Endocytosis is a fundamental biological process with numerous links to human diseases. We have been performing a genome-wide siRNA screen to identify regulators and novel components of two endocytic pathways. For this purpose, we developed a multi-parametric three-colour assay and pushed our homemade image analysis solution Motion Tracker II towards extracting over 60 different parameters. We screened 4-7 siRNAs per target of different sources’ libraries and compared those to a library of endoribonuclease-prepared esiRNAs. The degree to which different single siRNA oligonucleotides designed to silence the same genes actually mirror each others phenotype across the multiple parameters is an exceptionally sensitive means of establishing the specificity and extent of off-target effects. Screening data will be presented, and challenges and limitations of siRNA technology in correlation to truly multi-parametric assays will be discussed.

9:05-9:30 Automated High-Content Genome-Wide Screening Using Cellular Microarray Imaging
To be Announced

10:45-11:00 New Technology Presentation
Cytotoxicity Assays for High Content Screening
Stella Redpath, PhD., Group Product Manager, Drug Discovery, Millipore

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 (Opera^TM, 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.

10:45-11:00 New Technology Presentation
Harnessing the Power of RNAi and HCA Through Their Application to Discovery Research
Thomas Murphy, Ph.D., Dharmacon RNA Technologies, Thermo Fisher Scientific
Successful merging of RNA interference (RNAi) and high content analysis (HCA) requires the identification of critical attributes that distort the outcome of gene silencing studies. Data presented will demonstrate how combining these technologies with HCA can be utilized to understand the processes regulating stem cell differentiation and transcription factor activation.

11:00-11:25 Discovery of Novel Anti-Malarial Targets by Cell-Based High-Content Analyses Using siRNAs, Synthetic Compounds and Antibodies, Followed by in Vivo Validation in a Rodent Model
Christophe Echeverri, Ph.D., CEO, Cenix BioScience GmbH
We have used a focused library of siRNAs, combined with a high-content microscopy-based assay for Plasmodium infection in human hepatoma cells to screen through the entire kinome and investigate the role of lipoprotein pathway components in the liver stage of malaria infection. Several strong positive hit candidates were identified, including scavenger receptor BI (SR-BI), the major liver receptor for HDL. The SR-BI loss-of-function phenotype was consistently observed with multiple distinct siRNAs, and further confirmed using synthetic compounds and a blocking antibody, all previously shown to inhibit SR-BI function. These findings from in vitro cell-based work, combining automated image analyses of parasites inside the host cells with a complementary FACS-based readout, were also supported by in vivo analyses, including systemic siRNA treatments and genetic knock-outs, thereby validating the power of our HCA/HT-RNAi-based approach for discovery.

11:25-11:50 Integration of High-Content Imaging with Automated Genomic siRNA HTS for Novel Therapeutic Target Identification and Validation
Laszlo Kiss, Ph.D., Research Fellow, Automated Biotechnology, Merck & Co., Inc.

3:25-3:50 Use of HCA for Translational Safety Biomarkers
Peter O’Brien, Ph.D., Veterinary Clinical Pathologist, University College Dublin
High-content analysis (HCA) of morphological and biochemical parameters of live, cultured human cells has been demonstrated to be concordant with human toxicity potential of drugs. Accordingly, HCA may provide translational safety biomakers for drugs with such potential, especially with anti-infectious and anti-cancer chemotherapies. Application of HCA to cells circulating in the blood may enable early detection and monitoring of off-target subcellular effects, for example on mitochondria, lysosomes, cell proliferation, and oxidative stress. This presentation will address the preceding and provide some preliminary supporting data.

3:50-4:15 Application of High-Content Early Toxicity Screening Assays for Lead Candidate Selection
Madhu S. Mondal, Ph. D., Head, Early Toxicology, Preclinical Safety Profiling, Novartis Institutes of BioMedical Research
Drug induced toxicity is one of the major reasons for withdrawal of approved drugs from the market. In pharmaceutical industry, drug toxicities significantly contribute to the project termination at various preclinical and clinical stages. The ‘old-school’ strategy of derisking drug toxicity effects is that the compounds should undergo safety-testing at a frequency of one compound at a time, during the latter phase of drug discovery when a single compound is identified for further development. While this standard evaluation process has continued to help bring new drugs to the market, more recently it has become clear that the late-stage failures due to toxicities are very expensive, both in terms of cost and time. One way to address this cost and time issue is to develop high-throughput in vitro assays that can predict toxicity potential of early-stage compounds. Here, the idea is that in vitro assays, if amenable to high-throughput automation, if predictive for follow-up testing, and if this can be run using small quantity (1-2 mg) of compounds, are well-suited for addressing the safety-liabilities of the early-stage research compounds. Given that a large number of compounds can be screened this way, a complete series of molecules, not just a single compound as it is normally done, can be tested using this approach. In our attempt to address the toxicity issues early, we have developed a number of early toxicity screens for our drug discovery programs. One example of such an assay is the High-content micronucleus (HCMN) assay. This assay can be aligned well with research-phase drug discovery projects. In this presentation, we will discuss alignment of HCMN and other early toxicity assays, as they apply to early-phase drug discovery programs.

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.

5:35-6:00 Assessing Cancer Therapeutics in a Large Breast Cancer Cell Line Panel Using High-Content Analysis
Nicholas Wang, Ph.D., Scientist, Life Sciences, Lawrence Berkeley National Laboratory
Our lab has gathered a large cohort of breast cancer cell lines that represent the molecular spectrum of disease seen in tumors. Using the panel, we have tested several dozen therapeutic agents for proliferative and apoptotic responses. The combination of high-content data measuring response rates coupled with molecular profiling data, has allowed us to begin to dissect markers for both resistance and sensitivity to these agents. This may allow for a more targeted therapeutic regimen for patients in the clinic and can also be used as a screening tool for clinical study design.

Advances in Fluorescent Probes and Biosensors

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.