a

Day 1 - Monday, September 20, 2010

7:00

Registration & Breakfast

7:55

Welcome and Opening Remarks

[KEYNOTE PRESENTATION]
 

Applications of SMRT™ Sequencing Outside the Performance Envelope of First and Second Generation Sequencing

Stephen Turner, Ph.D., Founder & Chief Technology Officer, Pacific Biosciences

Despite their nearly universal use in DNA sequencing for several decades, DNA polymerases possess performance characteristics far beyond what first and second generation sequencing technologies have achieved. Through the use of phospholinked nucleotides, which can be incorporated with near-native kinetics by DNA polymerases, and zero-mode waveguides, which allow single-molecule detection at the high nucleotide concentrations required by native DNA replication, the natural power of these enzymes has been harnessed by Pacific Biosciences for single-molecule, real-time (SMRT™) DNA sequencing, which exhibits long read lengths, high speed and thus fast time to results. The high processivity of certain phage polymerases is translated into long readlength and applied to the shotgun whole genome assembly of a hydrogen-producing bacterium, Rhodopseudomonas palustris (R. pal). To demonstrate the effectiveness of long reads and strobed reads in resolving complex genome structure, we employed a hybrid assembly strategy using Pacific Biosciences’ long reads and strobe reads in conjunction with reads from the Illumina sequencing platform. The resulting assembly produced significantly fewer contigs and longer contig lengths than the starting Illumina assembly, with no misassembles when compared to the finished genome sequence. We show the role these assemblies play in the network analysis of hydrogen production by R. pal. Because of the kinetic information that accompanies the primary sequence extracted with every read, it is possible to detect the presence of modified nucleobases in DNA strands. In addition to allowing direct detection of epigenetic marks such as methylcytosine, it also provides a means of performing hypothesis-free investigation of structural modifications to DNA bases at the whole-genome level. The fast time to result is ideal for analysis of viral genomes in the context of outbreaks of infectious disease. To demonstrate the agility of the system we apply SMRT sequencing to raw samples taken from influenza patients to analyze the strains present at the single molecule level.

Session I: Cell Based Assays
Moderator: Robert Damoiseaux, Ph.D., UCLA 

Breaking New Ground in High Throughput Screening

Robert Damoiseaux, Ph.D., Scientific Director of the Molecular Shared Screening Resources at the California Nano Systems Institute, UCLA

High Throughput Screening has made its evolution from a pure drug discovery tool into a methodology which can be applied to almost any scientific arena. While areas such as Functional Genomics with their RNAi and cDNA technologies are well known applications of HTS, HTS still holds hidden treasures and opportunities for breaking new ground as well as crossing over into disciplines such as nanotechnology, engineering and biofilm research. In this talk I will present three examples of how HTS enabled breaking new ground in drug discovery as well as applied science.

     ·  Radioprotective drugs which protect from the effects of ionizing radiation have applications in radiation disaster mitigation as well as everyday medical radiation oncology where patients receive high doses of radiation as part of their ongoing cancer treatment plan. Our high throughput screening efforts for compounds with radio-protective properties in a cell based format yielded compounds which have efficacies of more than 90% rescue from a lethal dose of radiation in the whole animal model.

     ·  Nanotechnology has the potential to provide answers to many of mankind’s most pressing needs such as energy and medical needs. While there is an explosion of novel engineered nano-materials which have made their way into consumer products from cosmetics to tires it remains unclear what the potential toxicological and environmental ramifications of the use of nanomaterials are. Here, HTS enables the toxicological characterization of nanomaterials at a pace which is able to keep up the development of novel nanomaterials. Examples of how HTS allows for flagging of problematic nanomaterials early on in the product development cycle and thus enables re-design with enhanced safety features will be presented.

     ·  Biofilms can be broadly defined as a mixture of microorganisms and extracellular polymeric substances attached to a surface of biological, man-made or natural origins and have been associated with major medical problems such as implant rejection or contamination of drinking water.  Our development of a high content screening platform for biofilm formation and removal provides a rapid, reliable and quantitative assessment of biofilms for drug discovery as well as surface engineering purposes. HTS allowed us to isolate some of the governing factors in biofilm formation on engineered surfaces such as e.g. surface hydrophobicity which we were able to correlate directly with biofilm adhesion.

A High Content Screening Approach for the ABCG2 Transporter: Merits of Live Cell Assays

Irena Steele, Senior Research Scientist, Memorial Sloan-Kettering Cancer Center

Our group has developed a live cell based assay to screen for modulators of the ABCG2 transporter using an isogenic pair of U87 cell lines with one cell line over expressing the transporter and used for screening and the other line used to assess fluorescence enhancement accumulation due to the physico chemical properties of identified modulators and to eliminate them as auto-fluorescent compounds. The assay was validated in a pilot screen against a library of 6,912 compounds including FDA approved drugs and has identified several chemical classes of modulators. I will present our findings and discuss the potential application of this approach for the elucidation of the ABCG2 function and regulation.

Cellular Nanomechanical Profile as Cancer Biomarker

Jianyu Rao, M.D., FCAP, Professor of Pathology & Epidemiology, UCLA Ronald Reagan Medical Center

Cellular mechanical properties, including cell elasticity, softness, adhesion, etc., can be quantitatively measured by nanotechnologies such as Atomic Force Microscope. Such mechanical profile may provide a novel class of biomarker that can be used as either prognostic marker or predictive marker for therapy. The aggressive tumor cells may be softer and less stiff and thereby have more flexibility and mobility than benign cells. The cell softness/stiffness is regulated by signal pathways of cytoskeletal remodeling. Using Atomic Force Microscopy, we recently measured the metastatic tumor cells obtained from remnants of patient’s body cavity fluid samples, and found a marked difference in softness between malignant cells and benign mesothelial cells present in the same sample, and common modulus of cell softness in different cancer types metastatic to body cavity (Nature Nanotechnology, Dec., 2007). We further demonstrated that chemopreventive and therapeutic agents including green tea extract and other commonly used chemotherapeutic drugs have profound effect on cancer cell mechanics. Works on primary tumors collected by fine needle aspiration and other cytological samples for example urine is now undertaking. Together, nanomechanical analysis can be performed on clinical cytological specimens, and Youngs modular E may be a quantitative functional biomarker that can be used for personalized cancer management. Cellular nanomechanical analysis combined with molecular expression may provide a new platform for cancer biomarker development.

Networking and Refreshment Break

Capitalizing on High-Content Screening Capabilities to Drive GPCR Drug Discovery

Andrea Weston Ph.D., Senior Research Investigator, Bristol-Myers Squibb

G-protein coupled receptors (GPCRs) represent the largest superfamily of membrane proteins in the body, responding to a variety of stimuli and mediating a diverse range of physiological functions. Given their versatility and their widespread expression thoughout the body, GPCRs have long been considered and proven to be attractive targets for therapeutic intervention. Classically, GPCRs are best-known for their ability to initiate a signaling cascade via heterotrimeric G proteins. However, this prevailing model has expanded considerably over the past decade, and it is now well-accepted that GPCRs can signal independently of G proteins, through β-arrestin. This and other additional layers of complexity have major implications in efforts to pharmacologically modify GPCRs, leading to the development of new approaches to study GPCR function and to screen for small molecule modulators. High-content screening (HCS), the automation of cellular microscopy coupled with image analysis, is a platform that is particularly well-suited for GPCR drug discovery efforts; owing largely to the combined ability to multiplex, monitor kinetic events, and examine subcellular structures. We have evaluated and used high-content assays both for GPCR hit identification and to further characterize hits identified by using more traditional platforms including Aequorin, FLIPR, and cAMP assays. The HCS platforms we have used include assays monitoring GPCR internalization, β-arrestin redistribution, and Erk activation. The results of these experiences will be outlined as well as our perspective on the value of HCS for GPCR drug discovery efforts.

Benefits:
- Hear about BMS experience with high-content platforms in GPCR hit-ID efforts
- Hear about the utility of HCS to support efforts to identify allosteric modulators
- A comparison of high-content approaches with more traditional GPCR screening platforms will be described
- A brief description of the hurdles overcome to enable ultra-high-throughput HCS will be provided
 

High-Throughput, Label-Free Screening Small Molecule Compound Libraries for Protein-Ligands in Drug Discovery Using Small Molecule Microarrays and a Special Ellipsometric Scanner

Xiangdong Zhu, Professor, Department of Physics, University of California, Davis

Small-molecule compounds including peptides remain a major source of therapeutic and preventative drugs. The first step in developing new small-molecule drugs or new applications of existing drugs against a protein target requires screening large libraries of small molecules with diverse structures and compositions for a small set of those that exhibit sufficiently high affinity to the target and have a suitable orthosteric or allosteric inhibition effect before further structural optimization and other developmental work. Since the number of small molecule compounds in a library is large, from tens of thousands to millions depending on the library, high-throughput screening (HTS) methods are needed. Small-molecule microarrays (SMM) on a solid support in combination with a binding assay platform are one of the viable HTS options.

We demonstrate that by combining an oblique-incidence reflectivity difference (OI-RD) scanner with SMM we can screen by binding affinity constants 10,000 small-molecule compounds on a single glass slide against a protein probe without fluorescence labeling. This label-free, high-throughput binding assay platform promises screening 50,000 ~ 75,000 compounds in one day under a single assay condition. With a number of such systems in operation, we should be able to determine equilibrium dissociation constants of a protein probe against 50,000 to 75,000 small molecule compounds in one day.

Identification of Synergistic Drugs Using Combination High-Throughput Screening (cHTS)

Richard Rickles, Ph.D., Director, Oncology, Therapeutic Area Research, CombinatoRx

Cancer monotherapies are frequently suboptimal as tumor cells have a tremendous capacity to evade the effects of drugs by mutation and/or pathway adaptation. Focus on the coordinated action at multiple molecular targets may provide unique therapeutic benefit not achievable with the “one-drug, one-target” paradigm. We have developed a proprietary combination high throughput screening platform (cHTS) to rapidly identify novel drug synergies of potential clinical utility. In this approach, we generate a dose matrix response for each chemical combination, capturing the combined activity of two or more compounds over a broad range of single agent concentrations to identify potency shifts and efficacy boosts that result in increased, selective killing of tumor cells. Thousands of combinations can be screened across a cell line panel of varying genetic backgrounds within weeks. Follow up analysis of interesting combinations in dozens of cells lines can be performed within a few months and provide mechanistic insight into target-relevant network biology that has the potential to be leveraged clinically. Such information can inform preclinical development paths as well as potential patient sub-populations that may respond favorably to particular drug combinations. We will describe our use of cHTS to identify novel enhancers of glucocorticoids for treatment of hematological malignancies. We will also present work on the analysis of PI3K and MEK inhibitor combinations in solid tumors. Combination drug discovery and clinical deployment is an essential initiative that can alter cancer treatment and improve patient outcomes.

IonFlux – Automated Electrophysiology with Plate Reader Simplicity

Cristian Ionescu-Zanetti, Chief Technology Officer, Fluxion Biosciences

Automated patch clamp (APC) addresses a need for high throughput screening of chemical entities altering ion channel function. Systems that can produce pharmacologically-relevant data rapidly and consistently find considerable utility in the pharmaceutical industry, but also increasingly in academic laboratories. Here we present data obtained with a novel APC platform utilizing a well-plate microfluidic design. Unlike existing devices, the IonFlux system offers continuous recording from cell ensembles during rapid solution switches in a small, bench-top footprint that resembles a conventional plate reader.
True whole cell voltage clamp was applied to linear arrays of up to thirty cells in parallel utilizing fully-featured 16 or 64 channel voltage-clamp amplifiers under computer control. Laminar flow of solutions in a microfluidic network delivered cells in suspension to the recording sites and enabled fast exchange of bathing solutions via an electro-pneumatic interface on either 96- or 384-well plates. Electrophysiological characterization was achieved for hERG potassium channels and the NaV sodium channel family, as well as extensive data on ligand gated channels (GABA-A and Alpha 1 nicotinic). Data shows the voltage-dependence of these currents and their block by pharmacological agents other assay formats. With this system, long duration continuous recording and microfluidics combine to produce exceptionally fast application and washout of candidate drugs. The incorporation of multiple experiments per well-plate enables many investigations to be performed in parallel, with multiple compound concentrations that are applied to each individual cell ensemble recording array. High throughput recordings can be made with enhanced reliability; data is presented for recording success, throughput and assay reproducibility.

Benefits:
1. Hear a full update regarding the next generation ion channel screening technology from Fluxion
2. New data will be presented showing recording from ligand-gated ion channels with unparalleled success rates and number of compound applications per unit time.
3. See how the first APC system to have a plate reader format enables a modular approach to ion channel screening, integrating seamlessly into existing robotic solutions and high throughput lines.

Lunch

Session II: Advance Methods in Drug Discovery
Moderator: Kartik Narayan, Merck

Exploring Ciliogenesis via HTS-RNAi

Pedro Aza-Blanc, Ph.D., Director, Functional Genomics Resources, Sanford|Burnham Medical Research Institute

Primary cilia are evolutionarily conserved cellular organelles that organize diverse signaling pathways. Genetic screens in model organisms have discovered core machineries of cilium assembly and maintenance. However, regulatory molecules that coordinate the biogenesis of primary cilia with other cellular processes, including cytoskeletal organization, vesicle trafficking and cell-cell adhesion, remain to be identified. A RNA interference screen was performed to identify human genes involved in ciliogenesis control. The screen identified 36 positive and 13 negative ciliogenesis modulators, which include molecules involved in actin dynamics and vesicle trafficking. Further investigation demonstrated that blocking actin assembly facilitates ciliogenesis by stabilizing the pericentrosomal preciliary compartment. Moreover, knockdown of modulators that are involved in the endocytic recycling pathway affected ciliogenesis.

New Usage of Protein-Compound Docking in Structure Based In-Silico Drug Screening

Yoshifumi Fukunishi, Ph.D., Senior Research Scientist, National Institute of Advanced Industrial Science and Technology

Two new usages of protein-compound docking will be introduced. One application is a selection of screening results obtained from ensemble docking. The other is a protein-ligand pocket prediction. The former method uses a set of drug-like compounds to select reliable in-silico drug screening results. If some active compounds are known, the screening results that rank these active compounds at the top should be reliable. If no active compound is known, how to select the result is in question. We proposed a concept of a set of “universal active probes” (UAPs), which is a set of small active compounds those bind to different kinds of proteins. The hit ratio of the true active compounds in in-silico screening showed positive correlation to that of the UAPs. Thus, if the UAPs were added to the compound library, the screening result that shows a high hit ratio of the UAPs could give reliable actual hit compounds for the target protein. The latter method (ProbeSite) uses a set of randomly selected compounds. The ligan-binding pocket can be found by the average value of the docking scores of the randomly selected compounds.
(1) The UAP method enables you to select the most reliable result from many in-silico screening results obtained from the ensemble docking.
(2) The UAP method enables you to select the most reliable 3D model from many 3D models of a target protein.
(3) The UAP method is not time-consuming.
(4) ProbeSite can predict ligand-binding pocket at high precision.

Advanced 3D and Ex Vivo Modeling for Biomarker and Drug Discovery

Brett Hall, Ph.D., Global Lead: Ex vivo & 3D Modeling, Janssen Pharmaceutica, a Division of Johnson & Johnson

How does the tumor microenvironment (TME) impact drug development? The TME can be defined, in general terms, as the dynamic and complex cellular and non-cellular environment that tumor cells experience within their natural host. It is now established that the complexity, structure and state of a given TME can significantly influence fundamental processes in cancer biology. In fact, a growing body of literature has demonstrated that the TME can impact tumor cell growth, survival, gene expression, cell epigenetics, physiologic response to paracrine/autocrine factors, cell:cell interactions, cell:extracellular matrix (ECM) interactions, motility, metastasis, invasion, tumor-like signaling networks, sensitivity/resistance to cytotoxic drugs, tumor metabolism/Warburg effect and tumorigenicity. With this knowledge, it is clear that we can no longer ignore the TME-related shortcomings of our primary preclinical models during drug development.

Networking and Refreshment Break

Session III: Novel Assay and Screening Technologies – Part 1
Moderator: Kartik Narayan, Merck

Cell-based Assay Miniaturization and Automation at GNF: Screening and More

Daniel Sipes, Director of Advanced Automation Technologies, Genomics Institute of the Novartis Research Foundation

At GNF, we recently expanded the utility of our proprietary automated platforms for cell-based screening more deeply into the drug discovery process. We have utilized GNF-built automation, as well as other commercially available technologies, to enable cost effective and rapid cell-based compound profiling. In addition, this approach has enabled scientists to run primary cell assays on a scale not otherwise practical.

Optimizing Value from HTS

Robert Bostwick, Ph.D., Director, CNSP HTS Center, AstraZeneca Pharmaceuticals

HTS has traditionally been used as a random brute force approach to finding new chemical assets for lead generation wherein a target is screened using a single assay optimized for high throughput and low cost and compounds are selected based on potency/affinity and cluster size. Historically, this approach results in about a 50% success rate in identifying useful hits. By analyzing HTS data in combination with information gained from other approaches such as computational modeling, fragment screening, and multi-parameter screening, the success rate in finding quality hits can be enhanced. Examples of these different combination approaches will be given.

Assays for siRNA Conjugated Antibody

Gloria Meng, Ph.D., Senior Scientist, Technology, Genentech

RNA interference induced by short interfering RNA (siRNA) has therapeutic potential, but delivery is a formidable task. Antibody-siRNA is being tested as a method for siRNA delivery. Sensitive assays are needed for pharmacokinetic studies. Developing assays for siRNA is challenging since it has only 21-23 nucleotides, too short for PCR. We compared two methods to lengthen the siRNA: adding poly A or a stem-loop primer to the 3’ end of the anti-sense strand. The stem-loop primer method was easier to perform. Using this method and an antigen capture step, we developed an Immuno-RT-PCR method that is suitable for measuring intact antibody-siRNA in serum.

Networking Reception and Poster Session

Top of the page

Day 2 - Tuesday, September 21, 2010

Continental Breakfast

Chairman's Review of Day One

[KEYNOTE PRESENTATION]
 

An Evolving Role for Academia in the High Risk Activities of Early Drug Discovery

Michael R. Jackson, Ph.D., Vice President, Drug Discovery and Development, Burnham Medical Research Institute

Many pharmaceutical and biotechnology companies are changing how they fill their drug pipelines, in recent years they have trimmed their early discovery efforts and focused their resources on processes further downstream. This reduced effort does not reflect a lower demand to discover new classes of drugs to treat serious diseases, rather a change in business model. Fortuitously, while industry has been scaling back, a number of academic institutions, like the Sanford-Burnham Medical Research Institute have with the encouragement of the NIH been scaling up early drug discovery efforts opening state of the art high-throughput screening and chemical genomics centers. Armed with these and other capabilities they are now able to take projects much beyond target identification and to identify compounds that “chemically validate” a target and thereby jump start the process by which basic research discoveries are translated in to treatments. The potential of uHTS of both small molecule as well as siRNA libraries especially in phenotypic assays (e.g. image based high content screens) is also starting to be realized. While technically challenging, such screens are identifying new targets/pathways in complex biology/disease. HCS screening is also beginning to play a key role in stem cell biology, allowing conditions to be identified that differentiate cells to a desired cell type as well as enabling “disease in a dish” screens.
As new partnerships between academia and industry are established, institutes armed with high throughput chemical biology are well placed to play a central role in the newly emerging model for drug discovery.

Session IV: Assays for Protein Therapeutics and Beyond
Moderator: Ruchira Dasgupta, Adnexus

Tools and Techniques for Discovery of Adnectins, A Novel Class of Targeted Biologics

Ruchira Dasgupta, Associate Director, Adnexus, a Brtisol-Myers Squibb R&D Company

AdnectinsTM offer numerous potential advantages compared to traditional targeted biologics, including speed of discovery, ease of manufacturing, and the ability to create multi-functional targeted products. Adnectins are generated using Adnexus’ proprietary protein engineering system, PROfusionTM. With PROfusion, Adnexus scientists can simultaneously engineer trillions of Adnectin protein variations at a time in order to efficiently identify, evaluate and optimize product candidates. The presentation outlines Adnexus’ approach in applying PROfusion, as well as automation and screening techniques, to the Adnectin discovery process.

In Vitro Screening Assays for Aptamer-cell Surface Receptor Interactions

Chris Dimitri, Ph.D., Senior Scientist, Archemix

Aptamers are nucleic acid macromolecules formed by intramolecular interactions within the chain of nucleotides. The complex three-dimensional shape of the macromolecule allows the aptamer to bind tightly against the surface of its target molecule. Aptamers are obtained through iterative rounds of selection against target molecules. Typically, selection occurs in solution thus restricting the choice of target molecules to secreted factors or soluble recombinant Fc-fusion forms of cell surface proteins. A concern with solution based selection for surface associated proteins is that a cell surface expressed target may behave or fold differently in the native state compared to a recombinant Fc-fusion protein. To address this, we have incorporated the use of cell lines expressing the target of interest in both the selection and assay screening processes. This approach improved aptamer selection success for cell surface proteins. Aptamers obtained from this approach demonstrated improved affinity compared to the recombinant Fc-fusion protein. The availability of the cell lines also allowed for development of numerous cell based assays including flow cytometry to determine affinity of aptamers, assess the mechanism of action of the aptamers and whether lead molecules competed with available inhibitors for target binding. Development of cell-based selections and assays improved the success of selection for cell surface expressed proteins.

Intracellular Peptides: Novel Modulators for Drug Discovery and High Content Target Validation

Hanjo Henneman, CEO and CSO, Nexigen GmbH

Novel concepts and technologies are needed to target disease mechanisms, which are not accessible by common drug classes, like small molecules or antibodies. This applies specifically to target proteins, which are non-enzymatic and intracellular. Intracellular peptide therapeutics recently made their way into the clinic in several projects, and showed proof-of-concept in man.
The talk will focus on this novel drug class, and discuss technology to identify peptidic modulators targeting intracellular target proteins. It will be shown, that such peptide modulators can be used for the development of peptide derived drugs, but also for high content target validation.

- learn about the modulation of non-enzymatic target proteins by intracellular peptides
- hear why intracellular peptide drugs are a validated alternative to siRNA-therapeutics
- learn about novel peptide discovery technologies
- hear why intracellular peptides are excellent target validation tools, which are drug-like and generate high content data

Multiplexed Cell-Based Screening Assays by High-Throughput Flow Cytometry

Victoria Yatsula, Product Manager, IntelliCyt

High-throughput flow cytometry (HTFC) is a highly sensitive and quantitative method for performing multiplexed analysis of individual cells or beads - from binding assays to functional and mechanistic screens. The HTFC Screening System integrates the power of flow cytometry into a high-throughput screening platform that is capable of reading microplates at rates up to 40 wells per minute, such that a 384 well plate can be sampled in 10 minutes with sample uptake of 2μl.
This presentation will cover innovative, process-efficient and cost-effective screening tools for protein therapeutics that can be applied to antibody and peptide screens, including all stages of a screening cascade, from primary hybridoma or phage display library screens to lead evaluation functional assays. We will also discuss the use of endogenous versus recombinant screening cell lines, enabling more physiologically relevant therapeutic models.

Networking and Refreshment Break

[Oral Presentation from Exemplary Submitted Abstracts]

Characterization of an in vitro Differentiation Assay for Pancreatic-like Cell Development from Murine Embryonic Stem Cells: Detailed Gene Expression Analysis

Hsun Teresa Ku, Ph.D., Beckman Research Institute of City of Hope

Embryonic stem ES cell technology may serve as a platform for discovery of drugs that treat diseases such as diabetes. However, screening for molecules that regulate pancreatic beta cells and their immediate progenitors is limited thus far, because of difficulties in establishing reliable ES cell differentiation methods and in creating cost-effective plating conditions for the high throughput format. A relatively simple and inexpensive differentiation protocol that allows efficient generation of insulin-expressing cells from murine ES cells was previously established in our laboratories. In this report, this system is characterized in greater detail to map developmental cell stages for future screening experiments. Sequential activation of multiple gene markers for undifferentiated ES cells, epiblast, definitive endoderm, foregut and pancreatic endoderm, pancreatic progenitors, endocrine, exocrine, and ductal lineage cells was found to follow the sequence of events that mimics pancreatic ontogeny. Cells that expressed EGFP, driven by Pdx1 or insulin 1 promoter, correctly expressed known beta cell lineage markers. Day-20 cells implanted in vivo resulted in pancreatic-like cells. Overexpression of Sox17, an endoderm fate-determining transcription factor, at a very early stage of differentiation day 2-3 enhanced pancreatic gene expression. Overexpression of Ngn3, an endocrine progenitor cell marker, induced glucagon expression at stages when Pdx1 message was present day 10-16. Forced expression between days 16-25 of MafA, a pancreatic maturation factor, resulted in enhanced expression of insulin genes, Glut2 and glucokinase. Together, our differentiation assay recapitulates the proceedings and behaviors of pancreatic development, and thus will be valuable for future screening projects.

10:55

Electrochemical Impedance Microscopy: A Label-free Technique for Monitoring Individual Cells and Intracellular Processes

Wei Wang, Arizona State University

We present here a label-free and non-invasive electrochemical impedance microscopy EIM that can image the impedance of individual cells. Using EIM, we have studied many cellular processes, such as apoptosis, electroporation and drug-membrane receptors interactions, with sub-micron spatial resolution and millisecond temporal resolution. The basic principle of the EIM is based on the sensitive dependence of surface plasmon resonance SPR on surface charge density. We modulate the surface charge density of a SPR chip gold film by applying an AC voltage to the surface, then monitor the SPR response to the modulation.1 The amplitude of the SPR signal reflects the electrochemical impedance of the gold surface2, allowing us to image the surface impedance with high spatial resolution optically without detecting the electrochemical current. The technique allows us to measure the impedance changes of an individual cell during many cellular processes over a wide time scale, from seconds to hours. Simultaneously recorded EIM, optical and SPR images of the cellular processes provide rich information that cannot be obtained from other label-free techniques. Quantitative analysis of local impedance of individual cells and/or sub-cellular structures could be done by selecting different regions of interest of the EIM images. For example, EIM was used to continuously observe the apoptosis of individual cells in 8 hours. The results provided interesting sub-cellular impedance changes during the early-apoptosis stage. We have also applied the EIM to study fast cellular processes, including electroporation3 and drug-membrane receptors interactions which happened on a time scale of second. The EIM images reveal a large increase in the cell conductivity after the electroporation, and followed by a slow decay lasted for several minutes, which might reflect the formation and healing of membrane pores. Another example is to investigate the interactions between nicotinic acetylcholine receptors nAChR and their agonists like nicotine.4 Our EIM results indicates that the cell conductivity greatly decreases with the injection of nicotine, and the change is different at different sub-cellular locations, which can be attributed to the local distribution of nAChR in the cell membrane.

11:05

Global Analysis of Protein Kinase Activity During Hematopoietic Differentiation

Tyzoon K. Nomanbhoy, ActivX Biosciences

Cell proliferation and differentiation are highly regulated processes the deregulation of these processes can lead to a number of diseases including cancer. Protein kinases are key regulators of cell proliferation and differentiation, and therefore potential targets for a number of oncology indications. A detailed understanding of the changes in kinase activity/expression during cell proliferation and differentiation may be one of the keys to the identification of novel biomarkers and targets for the diagnosis and treatment of these diseases respectively. HL-60 cells are promyeloblasitc, and protocols for the differentiation of these cells into either macrophages by 12-O-tetradecanoylphorbol-13-acetate PMA, or granulocytes by retinoic acid RA are well-established. Importantly, HL-60 cell differentiation is a model system for myelocytic leukemia, and identifying the mechanisms that induce terminal differentiation of these cells may reveal effective targets for treatment of this disease. Toward this end, we evaluated the kinase activity and expression profiles of HL-60 cells throughout the differentiation process using desthiobiotin acyl phosphate ATP and ADP probes and liquid chromatography-mass spectrometry. Greater than 150 protein kinases in resting, RA, and PMA HL-60 cells were analyzed, and significant changes in activity were seen for a number of protein kinases in both differentiation pathways. These changes in kinase activity were generally consistent with changes in protein expression levels, as determined by Western blot. For both RA and PMA-induced HL60 differentiation, the kinetics for the changes in activity of the kinases was determined, and the results suggest that there is a well-defined sequence during which these changes occur. Using activity based protein profiling we identified a subset of kinases whose activity changed significantly during RA and PMA-induced HL-60 differentiation. Importantly, this methodology is widely applicable to other cellular systems and disease models, and could be applied in the identification of biomarkers and targets for the management of these diseases.

Session V: Novel Assay and Screening Technologies – Part 2
Moderator: William Taylor, Vertex Pharmaceuticals

Comparing the Throughput of Label-Free Real-Time Biosensors in Characterizing Large Panels of Monoclonal Antibodies

Yasmina Abdiche, Senior Principal Scientist, Pfizer

Label-free Methods for Fragment Screening

Kartik Narayan, Sr. Research Biochemist, Merck

SRU BIND – A Plate-based, Label-free Detection Platform for Cell-based and In Vitro Biochemical Applications

Eric Sandberg, Senior BioApplications Scientist, SRU Biosystems

Some drug targets are difficult to interrogate because of a lack of practical or valid biological assays. Label-free methodologies exist that can detect from simple binding events between small compounds and biomolecular targets to complex phenotypic changes in non-engineered cells. These plate-based, label-free biosensors are compatible with current drug discovery technologies, enabling a new approach to screen and profile intractable targets, as well as complementing or simplifying other methodologies.

SRU presents its BIND® technology, a robust, plate-based, universal assay system that enables label-free detection of biomolecular interactions. The system is comprised of the BIND Reader and 96-, 384- or 1536-well microplate BIND Biosensors. The BIND system takes advantage of a novel optical effect that generates very sensitive measurements of changes in binding or adherence in the proximity of the biosensor surface. This presentation will discuss BIND’s use as a drug profiling and screening tool for live cell-based and in vitro biochemical applications.

Analysis of functional cell response in live cells expressing GPCRs, RTKs and ion channels
• Robust and reliable measurement of endogenous cell targets
• Compatible with adherent and suspension cell types
• Information-rich data allows clustering of compound activity based on pathway mechanism
Direct binding biochemical assays
• Ideal for studying protein-protein interactions and other difficult to address targets

1. Learn about the newest additions to the suite of label-free instruments and microplates from SRU Biosystems, including our new high resolution, label-free Scanner
2. Learn how the Scanner is being used for stem cell studies, cell migration studies, and cell adhesion studies
3. See the newest customer-generated data in the areas of GPCR signaling, ligand-gated ion channel signaling, cell adhesion, cytotoxicity, and protein-protein interactions
 

Lunch

Tackling Challenging Targets with Fragment-based Approaches

Michelle Arkin, Associate Director, SMDC, University of California, San Francisco

The central tenant of chemical biology and small-molecule drug discovery is that biology can be manipulated using small, organic compounds. Nevertheless, the known drugs act on only ~1% of the proteome, and the realm of undrugged targets is vast. The aim of the Small Molecule Discovery Center is to develop drug-like compounds that advance our understanding of cell/protein function, validate promising new targets, and can serve as drug leads. We encounter a wide range of systems – from allosterically regulated enzymes to whole organisms – and have developed a similarly broad range of tools to address them. This talk will focus on our approaches to developing fragment-based ligand discovery for challenging and unusual targets, including enzyme allosteric sites and protein-protein interactions. We hope the audience will gain the following benefits:
• A perspective on building a fragment discovery center in an academic setting
• Case-studies on label-free technologies such as high-throughput SPR
• Approaches to “challenging” targets such as proteases, allosteric kinases, and protein complexes

Real-time Impedance-based Bioassays Using the xCELLigence Technology

Laura DeForge, Senior Scientist, Genentech

xCELLigence is a cell-based assay platform that allows for real-time, label-free monitoring of cellular responses by measuring electrical impedance. The 96 well assay plates contain electrodes that allow for measurement of the level of impedance of adherent cells in a readout termed Cell Index (CI). Changes in CI can be detected as a consequence of proliferation, cytotoxicity, or changes in morphology, adhesion, or migration. In our studies using cells transfected with a receptor tyrosine kinase (RTK), addition of ligand caused a dose-dependent increase in CI that could be inhibited by the addition of an anti-RTK antibody. The changes in CI measured by xCELLigence were found to correlate with changes in cell morphology as assessed by microscopy and with changes in the level of RTK phosphorylation as determined by immunochemical analysis of cell lysates. The xCELLigence technology was also found to perform well for other types of adherent cells and cellular responses.
Benefits of xCELLigence include:
• Real-time data collection over the course of the experiment yields more information than endpoint assays
• The readout is label free and does not interfere with cell growth or response
• Assay development is rapid, with minimal hands-on time with simple operation

The Relative Efficacy and Cost of Different HTS Active Followup Strategies

Bruce Koch, Director, Discovery Technologies, Roche

  3:00

Conference Concludes

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