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TRack II: Pathway Analysis

Focus Group:
High-Content Analysis for GPCRs

8:30-8:35   Chairperson’s Opening Remarks
Dr. Ralph Garippa, Research Leader, Roche Discovery Technologies, Hoffmann-La Roche, Inc.

8:35-8:50   High-Content Screening on a GPCR Target Platform
Dr. Ralf Heilker, Senior Scientist, Integrated Lead Discovery, Boehringer Ingelheim Pharma GmbH & Co. KG
The novel drug discovery technique, named “high-content screening” (HCS) by some vendors, has recently broadened the technology platform of the pharmaceutical industry for specific functional formats. In our opinion, 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. We intend to develop an HCS toolbox for compound profiling with regard to GPCRs.

8:50-9:05   Temporal and Spatial Aspects of Neurotensin Receptor Internalization
Dr. John Dunlop, Associate Director, Discovery Neuroscience, Wyeth Research
Agonist induced internalization of G-protein coupled receptors (GPCRs) is a well-established and nearly ubiquitous phenomenon generally believed to contribute to receptor desensitization. We have applied both confocal and high-content imaging techniques to establish assays to monitor neurotensin receptor internalization. These assays provide robust systems for studying dynamics and mechanism of agonist-induced receptor internalization.

9:05-9:20   Fully Integrated Robotic Approach Versus Workstation Approach: Two Norak Transfluor Screens and the Acumen Explorer HTS
Mr. Anthony Aglione, Associate Principle Scientist, Roche Discovery Technologies, Hoffmann-La Roche, Inc.
A systems based approach was taken to identify oGPCR’s for drug discovery HCS/HTS campaigns. Two of the identified oGPCR’s were developed and screened using the Norak Transfluor technology. One campaign was run utilizing a fully integrated Beckman-Saigen composite robotics system with an Orca robotic arm handler, while the other utilized an offline workstation consisting of two Tomtec Quadra systems. The Acumen Explorer HTS system was used to quantify receptor activation after a one hour incubation with compounds. We will discuss the advantages and disadvantages of both approaches with regards to throughput rate, time efficiency, material waste, curator handling, assay downtime and maintenance requirements.

9:20-10:00   Panel Discussion
Chairperson: Dr. Ralph Garippa, Research Leader, Roche Discovery Technologies, Hoffmann-La Roche, Inc. 
Panelists:
• Dr. John Dunlop, Associate Director, Discovery Neuroscience, Wyeth Research
• Dr. Ralf Heilker, Senior Scientist, Integrated Lead Discovery, Boehringer Ingelheim Pharma GmbH & Co. KG
• Mr. Anthony Aglione, Associate Principle Scientist, Roche Discovery Technologies, Hoffmann-La Roche, Inc.
Discussion Questions Include:
• What subsets of GPCRs are most amenable to HCS (such as orphans, Gi-coupled, Go-coupled receptors)?
• How does HCS compare to other screening technologies for Gq receptors (such as FLIPR or IP3 assays) and for Gs receptors (such as cAMP via luminescence, complement, or ELISA)?
• What is the rationale behind using b-arrestin reporter assay vs. directly tagged GPCR tracking?
• Is there any need or benefit to multiplexing in GPCR HCS assays?

Focus Group:
Integrating siRNA with HCA for Target Validation and Pathway Analysis
Sponsored By: 

8:30-8:35   Chairperson’s Opening Remarks
Dr. Steven Haney, Principal Scientist/Group Leader, Oncology Genomics, Wyeth Research

8:35-8:50   Apoptosis Assay to do Target Validation in Human Cancer Cells
Ms. Hong Xiao, Research Scientist, Applied Genomics, Bristol-Myers Squibb Company
A high-content screening approach was developed to study the apoptotic events in human cancer cell lines after siRNA treatment. The assay is highly sensitive and reproducible in capturing cells at several apoptotic stages. siRNA directed against genes that induce apoptosis show different responses in apoptosis parameters. Targets can be categorized by testing siRNA treatments into apoptotic profiles.

8:50-9:05   High-Content Screening for Target Validation Using siRNA
Ms. Shannon Hamilton, Senior Scientist, Roche Discovery Technologies, Hoffmann-La Roche, Inc.
High-content screening (HCS) is being used for target validation in early drug-discovery by defining the functions of genes and proteins as well as their signaling pathways. A family of proteins was treated with their specific siRNAs, then analyzed using a HCS mitotic index assay to determine which members could be potential drug discovery targets. Each potential target was evaluated for synergistic effects on both cellular function and gene expression using combinations of siRNAs in a matrix experiment. This presentation will show how the use of high-content screening enabled us to distinguish distinct morphological changes that were caused by the addition of the siRNA. In a second study of a relatively unknown protein, with few published literature references, we sought to elucidate effects on possible signaling pathways. Data defining the functional changes in mitotic index, NFkB translocation, and cell cycle detected on the Cellomics ArrayScan II will be discussed.

9:05-9:20   RNAi-Based Phenotypic Profiling Using High-Throughput, High-Content siRNA-Based Screens 
Dr. Christophe J. Echeverri, CEO, CSO, Cenix BioScience GmbH 
We have been combining high-throughput, genome-driven applications of RNAi using siRNA libraries with high-content assay methodologies in human cells, to achieve higher efficiency and patho-physiological relevance in our large scale target discovery and validation efforts. The use of multi-parameter readouts based on multiplexed microscopy-based assays is allowing more in-depth characterization of RNAi-induced loss-of-function phenotypes, enabling the RNAi-based phenotypic profiling of targets and compounds, and thereby offering new ways of advancing the discovery and development of new therapeutic compounds. 

9:20-10:00 Panel Discussion
Chairperson: Dr. Steven Haney, Principal Scientist/Group Leader, Oncology Genomics, Wyeth Research
Panelists:
• Dr. Christophe J. Echeverri, CEO, CSO, Cenix BioScience GmbH
• Mrs. Hong Xiao, Research Scientist, Applied Genomics, Bristol-Myers Squibb
• Ms. Shannon Hamilton, Senior Scientist, Roche Discovery Technologies, Hoffmann-La Roche, Inc.
• Dr. Nick Thomas, Principal Scientist, GE Healthcare 
Discussion Questions Include:
• What is the practical experience of multiparametric assays? Do they provide greater focus on hits or more hits? 
• Does the phenotypic response to siRNA correlate to the small molecule response directed at the same target? 
• How do you handle the statistical analysis of a biased sample set, for RNAi screens? Are hit rates too high?

Focus Group:
High-Content Analysis for Kinases

10:30-10:35   Chairperson’s Opening Remarks
Dr. Paul A. Johnston, Research Advisor, Eli Lilly and Co.=

10:35-10:50   High-Content Screening Approaches to Study Kinases
Dr. O. Joseph Trask, Jr., Associate Senior Biochemist, Eli Lilly and Co.
Studying modulation of the kinase signaling pathways using high-content screening instrumentation and software analysis has increased in academia, biotechnology and the pharmaceutical arenas over the past few years. Kinases are involved in a wide range of cellular processes including but not limited to cell signaling, cell migration, cell cycle progression, and differentiation. In this talk I will discuss approaches used in HCS technology to better understand the role of kinase activity in cell models and methods used to help deconvolute data associated with high-content information.

10:50-11:0   Evaluating Kinase Activity Using the Translocation Paradigm
Dr. Claudia Derian, Research Fellow, Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, L.L.C.
Many kinases translocate to different intracellular compartments upon activation, i.e. cytoplasm to plasma membrane or cytoplasm to nucleus. This presentation will describe a model system for monitoring kinase activity using a fluorescently-tagged kinase, which exemplifies both the spatial and temporal changes that can be monitored by HCA. Furthermore, the ability of this assay to reveal novel biologic effects through the use of activator/inhibitor combinations will be discussed.

11:05-11:20   Identification of novel compound classes with high throughput, high content pathway profiling
Dr Len Pagliaro, VP Bus Dev, BioImage AS
This presentation will describe kinase signaling pathway profiling with Redistributon assays to assess mode of action for small molecule hit compounds acting in the PI3K signaling pathway. These profiles can be used to rapidly identify the pathway segments in which test compounds are acting, and they are amenable to the rapid progression of compound classes with novel as well as conventional modes of action. The key role of pathway profiling in the identification of a compound class with potent anti-tumor activity will be described.

11:20-12:00   Panel Discussion
Chairperson: Dr. Paul A. Johnston, Research Advisor, Eli Lilly and Co.

Panelists:
• Dr. Claudia Derian, Research Fellow, Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, L.L.C.
• Dr. O. Joseph Trask, Jr., Associate Senior Biochemist, Eli Lilly and Co.

Discussion Questions Include:
• What are the advantages and disadvantages of phospho-antibodies to measure activity vs. total protein expression with GFP chimeras or antibodies?
• How can siRNA be used to validate kinase targets? Does knocking down one kinase protein affect the primary kinase activity or do compensatory proteins restore activity of the target protein?
• What stimuli should one use to activate the kinase pathway? Should mixed cell population that produce cytokines or other growth factors be used to emulate natural growth conditions? 
• How long should stimuli and compound be added? Should co-addition of compound and stimuli be used?
• Is live or fixed analysis more appropriate?
• What are the image analysis limitations? Do companies provide algorithms for multiplexing multiple kinases or other proteins?

Focus Group:
Neuronal Screening

10:30-10:35   Chairperson’s Opening Remarks
Chairperson: Dr. John Dunlop, Associate Director, Discovery Neuroscience, Wyeth Research

10:35-10:50   Second Generation HCS Assays for CNS Drug Discovery
Dr. Carmel Nanthakumar, Senior Research Scientist, Automated Imaging, Merck Sharp & Dohme
A new generation of HCS assays are being developed for CNS targets otherwise inaccessible to drug discovery. These include high information assays with triple parameter readouts, which permit simultaneous determination of compound mechanism of action and selectivity profiling using novel HTS techniques. Kinetic-based HCS assays using human neural stem cells and neuronal network cultures have also been developed using automated imaging platforms. These multiparameter HCS assays analyze complex cell behaviors to facilitate approaches to novel target discovery.

10:50-11:05   Enabling Chemical Genomics of the Nervous System Using High-Content Analysis
Dr. Stephen J. Haggarty, Harvard University, Eli & Edythe Broad Institute, Chemical Biology Program
Screening diverse collections of small molecules using high-content analysis will enable a chemical genomics of the nervous system. These efforts require an array of phenotype-based assays to be performed using neuronal cells and novel computational tools. I will discus our efforts in this direction using multiplexed luminescence- and image-based assays of neuronal signaling (post-translational modifications), morphology (axon and dendrite formation), and synaptic transmission (genetically-encoded probes.)

11:05-11:20   Screening for Factors Promoting Survival and Neurite Outgrowth in Retinal Ganglion Cell Culture
Dr. John Kerrison, Assistant Professor, Ophthalmology, Wilmer Eye Institute, Johns Hopkins Hospital
The objective of this study is to identify novel molecules and signaling pathways that promote survival and neurite outgrowth in purified retinal ganglion cell culture. It is reasoned that such molecules may have potential therapeutic benefit in restoring or preserving vision in patients with blinding optic nerve disease such as glaucoma, ischemic optic neuropathy, and optic neuritis. Experience with brain derived neurotrophic factor (BDNF) has demonstrated the success of this strategy. The specific aims are to screen proteins from an expression library derived from a retinal cDNA library as well as a publicly available library of compounds. Following identification of these molecules, further studies will be performed in order to elucidate their signaling pathways and therapeutic benefit in animal models of optic nerve disease.

11:20-12:00   Panel Discussion 
Chairperson: Dr. John Dunlop, Associate Director, Discovery Neuroscience, Wyeth Research

Panelists:
• Dr. Stephen J. Haggarty, Harvard University, Eli & Edythe Broad Institute, Chemical Biology Program
• Dr. John Kerrison, Assistant Professor, Ophthalmology, Wilmer Eye Institute, Johns Hopkins Hospital
• Dr. Carmel Nanthakumar, Senior Research Scientist, Automated Imaging, Merck Sharp & Dohme

Discussion Questions Include:
• What are the unique challenges in neuronal screening (compared with cell line screening)? 
• What assays are useful beyond neurite outgrowth? Receptor redistribution? 
• What are the challenges with phenotypic screening vs. target-based screening? 
• What are the challenges/opportunities with kinetic assays?