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Tuesday, 25 September 2007
7:30-8:30 Conference Registration
8:30-8:40 Welcoming Remarks from Conference Director
Julia Boguslavsky, Cambridge Healthtech Institute
HCA for Compound Screening
8:40-9:10 Case Study: Monitoring of Chemokine Receptor
Activation Using High-Content Screening
Ralf Heilker, Ph.D., Senior Scientist, Lead Discovery,
Boehringer Ingelheim Pharma GmbH & Co. KG
The novel drug discovery technique, named
"high-content screening" (HCS) by some vendors, has
significantly broadened the technology platform of the
pharmaceutical industry for specific functional formats. We
believe that the implementation of this technology is of strategic
importance in order to extend the scope of information provided to
the exploratory projects to support lead generation and lead
selection. In principle, the HCS hardware platform automates
fluorescence-based microscopic image analysis for multiplexed
localization of cellular components. Novel optical developments
have enabled a rapid and autonomous processing for a large number
of microtitre plates. Sophisticated object recognition algorithms
allow these HCS systems to perform automated image analysis on an
industrial scale. Multiplexing based on differently coloured
fluorophores allows us to collect additional levels of information
about primary and secondary effects of molecular targeted agents
in a cellular environment. In this case study, we have applied HCS
to study the activation and internalization of chemokine
receptors. The HCS approach will be compared to some more
classical HTS approaches.
9:10-9:40 Identification of HIV-1 Entry Fusion Inhibitors
Using a Homogeneous Competitive Cell-Based Binding Assay on
a High-Content Screening Platform
Koen Van Acker, Ph.D., Principal Scientist, Research &
Early Development, 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"
regions (HR1 and HR2) of the viral gp41 surface protein mediate
fusion of host cell and viral membrane, through the prerequisite
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-binding assay. This assay
was initially developed for flow cytometry-based read-out and was
later transferred onto a high-content screening system (Opera™,
Evotec Technologies). Both versions of the assay have comparable
quality parameters and similar sensitivity for peptide inhibitors
of HIV-1 entry. However, the HCS assay can be executed at higher
throughput and lower cost. Confocal imaging and multi-parametric
analysis allow for better handling of cytotoxicity, fluorescent
compounds, and other assay artifacts.
9:40-10:10 HCS Assays for Assessing Anticancer Drug
Combination Effects
Marcel B. Bally, Ph.D., Head, Advanced Therapeutics, BC Cancer
Agency
Anticancer drug combination effects are often
determined using a primary cell-based screen, a screen that
focuses on a single assay endpoint such as cellular metabolic
activity. This approach to assess combination effects is
inadequate and is not effectively predictive for combination
effects in patients. It is believed that HCS methods, which can
rapidly assess combination effects based on multiple assay
endpoints such as cell number, viability, apoptosis, cell cycle
and autophagy in a single assay, will be more predictive of
combination effects in the clinic.
10:10-10:40 To be Announced
10:40-11:30 Coffee Break with Poster and Exhibit Viewing
New Technology Showcase
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11:30-11:45 Get RAD-icle - Overcoming the Assay
Optimization Bottleneck in HCS
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Sponsored by: |
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Mark Collins, Ph.D., Marketing Manager, Cellular Imaging,
Thermo Fisher Scientific
Large-scale adoption of HCS and HCA methods
depends upon efficient assay development, this includes both the
biology (reagents, cell lines, etc.), but also to a very large
extent on optimizing the image analysis to create robust and
relevant measures of the biology. Using two case studies, we will
present Cellomics' unique approach to Rapid Assay Development.
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11:45-12:00 Multiplex Analyses of Nuclear Receptor Function by High Throughput Imaging
Michael A. Mancini, Ph.D., Associate Professor, Department of Molecular and Cellular Biology, Baylor College of
Medicine
Using high throughput microscopy (HTM), we have developed multiplex single cell models that enable an increasingly integrated view of estrogen (ER) and androgen receptor (AR) functions. In AR studies in
HeLa, we have quantified the cytological distribution and transcriptional activity of wild type and mutant ARs in response to ~30 compounds (known agonists, antagonists or endocrine disruptors). EC50 values for nuclear translocation and subnuclear speckling were markedly different, and for agonists, a general (but not universal) correlation between speckling and transcription was observed. Importantly, the link between agonist-induced speckling and transcription was AR expression-level-dependent, demonstrating that speckling, in the absence of expression level monitoring, could be misleading when assaying for transcriptional antagonists. For higher level multiplex
analsyses, we developed a stable HeLa cell line containing a visible, multicopy promoter array regulated by ER. HTM was used to quantified array size and associated reporter gene mRNA in response to agonists and antagonists. Time course studies with two agonists showed cyclical accumulation of reporter gene mRNA accumulation after
estradiol, while EGF stimulation provided a single pulse of transcription. Using a panel of antibodies to ER coregulators and multi-channel image analysis, we will discuss “visual ChIP” approaches to define promoter array occupancy that can be linked to chromatin changes and mRNA synthesis. In conclusion, we have developed multiplex single cell assays for rapid characterization of transcription function amendable to mechanistic dissection by high content screening.
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12:00-12:15 New Developments in HCS Screening Related to Brightfield, Automated User Defined Focus Points and Unique Data Mining Tools for Increased Data Quality and
Assessment
Kris Ver Donck, VP Technology, MAIA SCIENTIFIC
High Content Screening has mostly been focused on fluorescent applications. Current developments in Brightfield image acquisition and analysis, either as a stand-alone application or together with fluorescence applications, can provide a better assessment of cell functionality while reducing the number of staining procedures. Unlabelled live cell analysis, repeatable assays on the same cell cultures, clonal assessment and cell count related to these new developments are reviewed as well as the use of a user defined automated focus tool for abnormal cell structures. Additionally, a unique data-mining tool will be demonstrated for fast and intuitive data analysis.
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12:15-12:30 Technology Short
Talk
Additional Sponsorship Available. Please contact Carol
Dinerstein at 781-972-5471 or dinerstein@healthtech.com.
12:30-14:00 Lunch on Your Own
HCA for Toxicity and Efficacy Assessment
14:00-14:30 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
biomarkers 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.
14:30-15:00 Development of Cell-Based Assays for Study of
Cardiac Disease and Drug Efficacy and Toxicity Using Primary
Cardiomyocytes and Cardiac-Derived Cell Lines in a
High-Content Format
Anthony Davies, Ph.D., Director, High-Content Research
Facility, Department of Clinical Medicine, Trinity College Dublin
We are currently engaged in the development of
a range of cell-based assays, which utilize both primary adult
cardiac myocytes and immortalized cell lines. To facilitate the
development of these new assay tools, a detailed examination of
both biochemical and structural changes in primary adult cardiac
muscle cells has been conducted. These studies have yielded
valuable information regarding the behavior of primary cardiac
muscle cells in their quiescent and active states. Currently, our
work is focused on the use of muscle cells derived from a cardiac
myoblast cell line as a basis for primary and secondary cardiac
screens. Our ultimate goal is to develop a stable and biologically
relevant assay that can be deployed and utilized in an automated
HCS format.
15:00-16:00 Refreshment Break with Poster and Exhibit
Viewing
HCA for Mechanism-of-Action Studies
16:00-16:30 Use of the BD Pathway in CNS Drug Discovery
to Determine Mechanisms of Compound Action in Primary
Neuronal Cultures
Claire Scott, Senior Scientist, Molecular Pharmacology,
Neurophysiology & Pharmacology, Psychiatry CEDD,
GlaxoSmithKline
Functional data generated using recombinant GPCR-expressing cell lines does not always translate directly to
neuronal systems because of overexpression of transfected
receptors and\or inappropriate\promiscuous coupling to signal
transduction pathways not observed in the native tissue host cell
type. The 96 well plate BD Pathway single cell confocal imaging
system provides the opportunity to perform medium-throughput,
multiple end point, quantitative pharmacological analyses in
neuronal populations, thereby enabling realistic evaluation of
agonist\modulator trafficking of intracellular signals in the CNS.
16:30-17:00 Development of Automated Image Analysis
Routines for Functional HCA
Philip Denner, Ph.D., Lead Discovery Berlin, Screening,
High-Content Analysis, Bayer Schering Pharma AG
The development of assay-specific image
analysis routines is a critical issue. They have to be able to
identify, correlate and quantify specific intracellular signals. A
flexible programming interface enables the establishment of
specific image analysis routines which can measure kinase-dependent
substrate phosphorylation in the nucleus of eucaryotic cells and
subsequent substrate translocation into the cytosol. We used this
approach during secondary screening to determine the impact of
kinase specific inhibitors within the cellular context.
17:00-18:00 Cocktail Reception with Exhibit and Poster
Viewing
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