Wednesday, 26 September 2007

7:30-8:15 Breakfast Technology Workshop

Sponsored by

Quantitative Imaging Approaches in Drug Discovery Werner Stürmer, Ph.D., Head, High-Throughput Screening, Altana Pharma AG During the last few years, high-throughput imaging systems became available and may now be used in the early drug discovery process and also increasingly in the HTS field. Especially, by the use of confocal microscopy methods, quantitative imaging approaches became feasible, which currently are exploited only to a minor extent. This talk will give an outline on theoretical principles, describe experiences and challenges during implementation and, finally, present examples of quantitative, biochemical and cellular assays run in HTS mode.

10:15-10:45 Large-Scale siRNA Screening for Functional Genomics in Endocytosis
Martin Stöter, Ph.D., Scientist, High-Content Screening, HT-Technology Development Studio (TDS), Max Planck Institute of Molecular Cell Biology and Genetics
At the TDS, high-content cell-based assays are developed and RNAi technology is used for functional genomics. We use automated liquid handling procedures for transfecting and processing cells in a three colour secondary assay like large-scale RNAi screen. Automated high-throughput confocal microscopy provides high-resolution images for complex multi-parametric image analysis. A homemade image analysis solution in combination with high-power computing allows us to discriminate between ca. 60 different parameters. Different RNAi technologies and libraries are used to identify novel regulators and components of different endocytic pathways. The screening progress will be presented.

10:45-11:15 Identification of Novel Anti-Malarial Targets and Lead Compounds Using High-Content Microscopy Assay for RNAi and Drug Screens
Michael Hannus, Ph.D., Group Leader, Cell-Based Discovery and Validation, Cenix Bioscience GmbH
The asymptomatic liver stage of Plasmodium infection offers a unique intervention point for the development of prophylactic anti-malarial agents that can stop the disease before it ever reaches the devastating blood stage. We applied an RNAi based infection assay in human hepatoma cells to identify those human liver genes that are non-essential to the host but necessary for Plasmodium infection. Screening through a custom library of synthetic siRNAs, we identified Scavenger Receptor B1 (SR-B1) as a critical host factor for both host cell invasion and parasite development. The role of SR-B1 in Malaria was further investigated in vitro and in vivo.

11:15-11:45 A High-Content Assay Reveals the Potential Role of microRNA Clusters in Cell Growth and Transformation
Anja van Brabant-Smith, Ph.D., Assay Development Scientist, Dharmacon, Thermo Fisher Scientific
microRNAs (miRNAs) have been implicated in the regulation of a broad range of cellular processes (e.g. tumorigenesis, apoptosis, and differentiation), through the repression of target gene expression. Many miRNAs are transcribed from the genome as clusters that originate from a single common primary transcript (pri-miRNA), suggesting a coordinated mode of expression and regulation. However, the precise functions of miRNAs have been difficult to identify because the effects of inhibiting individual miRNAs appear to be subtle. To address the hypothesis that miRNAs may act in concert, we systematically inhibited a set of miRNA clusters using highly potent miRNA inhibitors in MCF7 cells, a breast cancer cell line. We used a high-content assay to monitor the effects of miRNA inhibition on a series of sub-cellular processes including the activation of c-Jun, translocation of NF-êB, and cell proliferation. We identified several clusters of miRNAs that, when inhibited, result in an increase in activated c-Jun, translocation of NF-êB, and/or a decrease in cell number. Our results support a role for these endogenous miRNAs in cellular growth and transformation.

11:45-12:15 Drugable Genome-Wide RNAi Screen in Huntington Disease Applying High-Content Analysis
Remko de Pril, Scientist, Neurodegeneration, Biofocus DPI
Huntington disease is an autosomal dominant neurodegenerative disorder for which no cure is currently available. Expansion of the polyglutamine repeat in the Huntingtin protein leads to aberrant folding and inclusion formation of the protein. Toxic subspecies of the aggregation prone protein are eventually resulting in neuronal death in patients. Applying Biofocus DPI’s adenoviral technology we developed a high-content screen to identify targets that inhibit polyglutamine induced toxicity. Mutant Huntingtin was expressed in SH-SY5Y neuroblastoma cells and we have now screened over 40,000 wells. We identified a high number of novel primary hits out of both our SilenceSelect ­shRNA as well as our FLeXSelect ­cDNA library. The assay showed a very high reproducibility and HCA additionally enabled us to study the mode of action of our hits. Interesting hits can consequently be selected that inhibit polyglutamine-induced cell-death by increasing the inclusion ratio, or inhibit even in the presence of elevated levels of mutant Huntingtin. We will demonstrate the results of what is to our knowledge the first full primary RNAi screen to be performed on a High-Content Platform and report on the advantages and hit-classes of our screen.