Wednesday, January 16

7:30-8:15 Technology Workshop
(Sponsorship Available. Contact Carol Dinerstein at dinerstein@healthtech.com or 781-972-5471)

8:30-8:55 Reports from ThinkTank Roundtable Discussions

Increasing Multiplexing in Screening

9:00-9:30 High-Content Simultaneous Analysis of Multiplexed Targets with Combinatorial Chemical Libraries
Larry A. Sklar, Ph.D., Regents Professor of Pathology and Distinguished Professor of Pharmacy, Director of Basic Research, UNM Cancer Research and Treatment Center; Director, New Mexico Molecular Libraries Screening Center
We are prototyping HTS approaches that use the multi-parameter optical capabilities of the flow cytometer for the discovery of active small molecules from compound libraries. We have been interested in two approaches to improve the efficiency of discovery by increasing biological and chemical content. Biological content is increased with mixtures of targets that are color-coded as suspension arrays. Several receptors or transporters on distinct cell populations, or several molecular assemblies on distinct microsphere populations are mixed in a single assay well. This approach has been successfully applied to GPCR receptor families, MDR transporters, low molecular weight G proteins, Bcl-2 family interactions, and substrate families for proteases. The deconvoluted data reveal small molecules with selectivity and specificity in both HTS primary screens and dose-response analysis. To increase chemical content, we have used mixtures provided by TPIMS of thousands of compounds per well arrayed in combinatorial fashion. After data deconvolution and resynthesis based on the predicted structures, individual small molecules with selectivity and specificity are revealed. Taken together, we have demonstrated proof of principle for simultaneous high-content analysis of biological and chemical space, using multiplex targets and combinatorial chemical mixtures and potential increases in content by many thousand fold.

9:30-10:00 Addressing Difficult Targets with Cellular Technologies
Jeremy S. Caldwell, Ph.D., Director, Molecular and Cell Biology, Genomics Institute of the Novartis Research Foundation
Post-genomic tools have aided in the identification of a significant number of critical effector genes and putative drug targets involved in controlling disease states. Unfortunately many of these targets are either not classically druggable (e.g. enzyme, surface receptor, etc.) or not amenable to current small molecule screening technologies. Recently a series of new cell-based strategies aimed towards identifying tools and lead drug molecules for this interesting set of targets have come to the fore. This talk will highlight these technologies, their applications to different target classes, and parallel advances in molecular biology and automation critical to their successful application.

10:00-11:00 Coffee Break with Exhibit and Poster Viewing

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

11:00-11:25 High-Content Screening to Identify Small Molecules Inducing Plasma Membrane Translocation Events or Neurotoxicity
Daniela Gabriel, Ph.D., Associate Director, Center for Proteomic Chemistry, Lead Finding Platform, Novartis Institutes for BioMedical Research
High-content screening is defined as the automation of high-content cell biological investigation of arrayed cells including key operations of experimental design, sample preparation, image acquisition, archiving, processing and analysis, and cellular knowledge mining. In the talk, plasma membrane trafficking projects tackling different mechanisms will be discussed. Furthermore, we established a neurotoxicity assay using primary cells in a medium throughput format to identify specifically neurotoxic small molecules. To improve the analysis of the assay, we analyzed the data with several image analysis modules for neurite outgrowth using programs from different vendors. The outcome of this comparison will be presented.

11:25-11:50 High-Content Microscopy Analysis of Ab_1-42-induced Neurite Outgrowth Reduction in Rat Primary Cortical Neurons: Neuroprotective Effects of a7 Neuronal Nicotinic Receptor Ligands
Jinhe Li, Ph.D., Research Investigator, Neuroscience, Abbott Laboratories
b-amyloid peptide 1-42 (Ab_1-42) is generated from amyloid precursor protein (APP) and associated with neurodegeneration in Alzheimer’s disease (AD). Ab_1-42 has been shown to be cytotoxic when incubated with cultured neurons. However, APP transgenic mice over expressing Ab_1-42 do not show substantial loss of neurons, despite deficits in learning and memory. It is thus emerging that Ab_1-42-induced memory deficits may involve subtler neuronal alternations leading to synaptic deficits, prior to frank neurodegeneration in AD brains. In this study, high-content screen (HCS) microscopy, an advanced high-throughput cellular image processing and analysis technique, was utilized in establishing an in vitro model of Ab_1-42-induced neurotoxicity utilizing rat neonatal primary cortical cells. Neurite outgrowth was found to be significantly reduced by Ab_1-42 (300 nM – 30 µM), but not by the scrambled control peptide control, in a time- and concentration-dependent manner. In contrast, no reduction in total number of neurons was observed. The Ab_1-42-induced reduction of neurite outgrowth was attenuated by NMDA receptor antagonist memantine and the a7 nicotinic acetylcholine receptor (nAChR) selective agonist PNU-282987. Interestingly, the a7 nAChR antagonist methyllycaconitine also significantly prevented reduction in Ab_1-42-induced neurite outgrowth. The observed neuroprotective effects could arise either from interference of Ab_1-42 interactions with a7 nAChRs or by modification of receptor-mediated signaling pathways. In summary, our studies demonstrate that reduction of neurite outgrowth may serve as a model representing Ab_1-42-mediated synaptic failure in AD, which, in combination of HCS, provides a high-throughput cell-based assay that can be used to evaluate compounds with neuroprotective properties in neurons.

11:50-12:15 Measuring Synaptic Function in Primary Neurons Using High-Content Analysis
Michael P. Bova, Ph.D., Principal Scientist, Target Advancement Group Leader, Dept. of Biology, Elan Pharmaceutical Inc.
In this study, we investigate the effect that Ab_1-40 as well as a panel of small molecule neurotoxins have on neuronal morphology and synaptic function in human cortical cultures. We will highlight Bioapplications from the Cellomics platform that will allow us to measure neuronal morphology and synaptic events. We observed that different classes of neurotoxins show differential effects on parameters of neuronal morphology. For example, phosphatase inhibitors had a much greater effect on branch points than on neurite length. In contrast, with 6-OH dopamine, all parameters of neuronal morphology were equally affected. We also explored the potential of using FM1-43 dyes to monitor exocytosis in primary neuronal cultures. The results of this study demonstrate that assays developed using HCA are well-suited to monitor neuronal morphology
and function.

LABEL-FREE SCREENING

12:15-12:40 Comparison of Cell-Based Label-Free Assay Tools
Lisa Minor, Ph.D., Principal Scientist, Vascular Biology, Johnson & Johnson
Measuring cellular events from exogenous cellular stimuli using a common detection technology without having to modify the cellular environment by addition of endogenous labels is a wish that has recently been realized. Label-free technologies based on electrical impedance or refractive index offers such scenarios. This presentation will describe and compare these technologies and their application to cell-based assays using the G-protein coupled receptor, Urotensin II, as an example.

12:40-1:05 Label-Free Screening of Fragment Libraries by SPR Imaging of Chemical Microarrays
Thomas Neumann, Ph.D., Head of Screening, Graffi nity Pharmaceuticals
Fragment-based screening has recently evolved into a promising strategy in drug discovery. In particular, surface plasmon resonance (SPR) imaging of chemical microarrays has been proven to be a powerful tool for primary screening of large fragment libraries. Small molecule libraries (110,000 compounds) are immobilized on gold chips to construct arrays with high spot densities (9,216 compounds/chip). Binding of soluble proteins onto such chemical microarrays can then be read out by SPR imaging in a label-free, function-blind and high-throughput fashion. The sensitivity of Graffinity´s SPR imager has shown to be well suited for the detection of even weak protein-fragment interactions with a minimum of target consumption. It thereby offers convincing solutions for rapid hit identification and array-informed hit optimization. Case studies demonstrate the successful identification of selective low molecular weight inhibitors for pharmacologically relevant drug targets.

1:05-2:00 Lunch on Your Own

2:00-7:00 BD Biosciences User Group Meeting

2:00-7:00 PerkinElmer/Evotec User Group Meeting

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