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  • Designed diversity and bioannotated compound libraries for obtaining maximal value from screens in induced pluripotent stem cell-derived cells and other complex biological systems

    Contains 1 Component(s) Recorded On: 02/06/2018

    ​As part of the current resurgence in phenotypic screening, early stage assay systems are becoming increasingly complex in an attempt to better model the disease state and therefore improve translation to the clinic. One downside to this complexity is that it substantially reduces the throughput of such systems, and it is generally not feasible to screen millions of molecules in them as would be common in a more traditional HTS campaign.

    As part of the current resurgence in phenotypic screening, early stage assay systems are becoming increasingly complex in an attempt to better model the disease state and therefore improve translation to the clinic. One downside to this complexity is that it substantially reduces the throughput of such systems, and it is generally not feasible to screen millions of molecules in them as would be common in a more traditional HTS campaign. In response to this constraint we have developed two focused compound collections for use in phenotypic screening projects. The first is a bioannotated or chemogenomic library containing molecules with well-characterised pharmacology, intended for use in repurposing and knowledge-driven target deconvolution approaches. The second is a diverse phenotypic library that attempts to maximise chemical and pharmacological diversity within a compact set of 20,000 compounds, intended as a representative sample of our regular HTS collection. In this presentation the design, construction and annotation of these libraries will be outlined. Some examples of our experiences with screening them in drug discovery projects will then be discussed. Finally, analysis of the extent to which these first generation collections have fulfilled their design criteria to date will be presented, and directions of future enhancements reviewed.

    Tim James

    Evotec

    With a background in computational chemistry and data analysis, my current focus within the Research Informatics group at Evotec is the area of chemical biology. This includes the design of compound libraries, the analysis of phenotypic screening results and integrating the output from various 'omics technologies.

  • Complex Tissue Biology and Throughput in a Microplate-Based Organ-on-a-Chip System

    Contains 1 Component(s) Recorded On: 02/06/2018

    In this presentation, I will focus on biological and technological aspects of both healthy and diseased tissue models in the OrganoPlate, including platform specific assays, such as a barrier integrity assay and neuronal network activity assays. I will present data demonstrating that 3D tissues cultured in the OrganoPlate are suitable for any-throughput drug efficacy and toxicity screening, trans-epithelial transport studies, and complex co-culture models in an in vivo-like microenvironment.

    The OrganoPlate is a microfluidic tissue culture platform, which enables high-throughput culture of microtissues in miniaturized organ models. In the OrganoPlate(1), extracellular matrix (ECM) gels can be freely patterned in microchambers through the use of phaseguide technology. Phaseguides (capillary pressure barriers) define barrier-free channels in microchambers that can be used for ECM deposition or medium perfusion. The microfluidic channel dimensions not only allow solid tissue and barrier formation, but also perfused tubular epithelial vessel structures can be grown. We have developed a range of multi-cellular organ- and tissue models for drug efficacy and toxicity studies, including blood vessels, brain, gut(2), and kidney.

    In this presentation, I will focus on biological and technological aspects of both healthy and diseased tissue models in the OrganoPlate, including platform specific assays, such as a barrier integrity assay and neuronal network activity assays. I will present data demonstrating that 3D tissues cultured in the OrganoPlate are suitable for any-throughput drug efficacy and toxicity screening, trans-epithelial transport studies, and complex co-culture models in an in vivo-like microenvironment.

    References:
    1. S. J. Trietsch, G. D. Israëls, J. Joore, T. Hankemeier, and P. Vulto, “Microfluidic titer plate for stratified 3D cell culture.,” Lab Chip, vol. 13, no. 18, pp. 3548–54, Sep. 2013
    2. S. J. Trietsch, E. Naumovska, D. Kurek, M. C. Setyawati, M. K. Vormann, K. J. Wilschut, H. L. Lanz, A. Nicholas, C. P. Ng, J. Joore, S. Kustermann, A. Roth, T. Hankemeier, A. Moisan, P. Vulto, “Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes.”, Nature Communications, 2017 accepted

    Jos Joore

    MIMETAS - the Organ-on-a-Chip Company

    Jos Joore is co-founder and Managing Director of MIMETAS. He is a biotech entrepreneur, co-founder of four companies with over 17 years of executive level biotech experience in a variety of companies including Pepscan, Skyline Diagnostics, Kreatech and Westburg. During his ten-year research career, he worked as a postdoctoral researcher at the Hubrecht Institute (Utrecht) and King's College (London). He holds a cum laude Masters degree in business marketing (MBM), a Ph.D. in developmental biology and a Masters degree in biology.

  • Lab Automation Drones for Mobile Manipulation in High Throughput Systems

    Contains 1 Component(s) Recorded On: 02/06/2018

    In lab automation, there is a wide range of robots. Robots are employed to accelerate sample handling, such as in high throughput screening (HTS), manipulators and transfer lines rapidly manipulate micro-plates amongst numerous test stations. The net result is that a typical HTS system can handle over 500,000 samples a week. In the age of big data, higher throughput means faster pharmaceutical development and hence quicker patent registrations and earlier market penetration.

    In lab automation, there is a wide range of robots. Robots are employed to accelerate sample handling, such as in high throughput screening (HTS), manipulators and transfer lines rapidly manipulate micro-plates amongst numerous test stations. The net result is that a typical HTS system can handle over 500,000 samples a week.  In the age of big data, higher throughput means faster pharmaceutical development and hence quicker patent registrations and earlier market penetration. HTS systems are often custom-tailored to maximize throughput with many high- precision 6-DOF robot manipulators. Such robots employ parallel jaw grippers to gently and precisely position and orient micro-plates. However, once configured, they are not easily changed. This is important because as new tests emerge, older HTS systems cannot easily perform them. The National Institutes of Health (NIH) in the United States are looking at the potential of lab automation drones to add flexibility to existing HTS systems. The notion has merit; aerial manipulation research is an active area. High degree of freedom (DOF) robots with dexterous arms has been addressed in transformative applications such as material handling, disaster response, and personal assistance. And micro-plates are relatively easy to robotically lift and orient. Issues like ground effect, limited battery life, and obstacle avoidance are indeed relevant to lab automation but also remain open research topics. The critical gap in a lab automation drone appears to be the lack of aerial manipulation arms and grippers. Recently, several configuration systems including single DOF aerial grasping, non-redundant and fully redundant articulated aerial manipulation, have been explored to create manipulation systems. But all the arms in aerial manipulation are serial; a motor in each joint results in a heavy arm. To the author’s best knowledge, the author’s lab has been the first to introduce a parallel-mechanism arm for aerial manipulation. The previous work concluded its higher degree of precision and lower toque impact on the drone’s stability versus serial manipulators. In this work, the authors present a design of a 6-DOF parallel mechanism arm with a sensorized parallel jaw gripper. The test-and-evaluation approach and results are given.

    Dongbin Kim

    University of Nevada, Las Vegas

    Dongbin Kim has completed his B.S. in aircraft systems engineering from Korea Aerospace University, S. Korea in 2016. He is currently studying PhD in Mechanical Engineering in University of Nevada in Las Vegas. He serves as lab manager in Drones and Autonomous Systems Lab.

  • Integrating Environmentally Friendly Tactics into a High-Throughput Screening Setting

    Contains 1 Component(s) Recorded On: 02/06/2018

    Throughout everyday life there are many considerations and practices in place when it comes to recycling, minimizing waste, cleaner energy and reuse to cut down on the impact to our planet’s ecosystem, with millions of individuals around the world making the choice to conserve keeping these principles in mind.

    Throughout everyday life there are many considerations and practices in place when it comes to recycling, minimizing waste, cleaner energy and reuse to cut down on the impact to our planet’s ecosystem, with millions of individuals around the world making the choice to conserve keeping these principles in mind.  However, this mindset and conscientiousness to conserve is not on the radar when it comes to the world of high-throughput screening and science in general, where the term ‘consumable’ is ubiquitous and pipette tips, petri dishes, microplates, solvents and an extensive list of materials are disposed of after one use every day, with most of this waste needing to be handled as chemical or biohazardous, further increasing the negative environmental impact.  Luckily this mentality is changing, with the availability of new technologies and the use of experimental data proving its effectiveness NCATS has been able to implement and adopt several methods into many aspects of their high-throughput screening processes which are friendlier to our environment than the traditional equivalents.  In many cases these eco conscious practices yield higher quality, cleaner data as well as even eliminating the need for automated assays having to be repeated by catching detrimental issues in real time.  Here the focus will be about the integration of equipment onto peripheral devices of the robotic screening platforms, processes and supporting modular operations with the overall goal of conservation.  Not only will the use of these concepts be demonstrated but more importantly the successful adaptation will be shown with supporting data.  Spanning the last 7 years NCATS has not only been mindful but has been continuously advancing and developing tactics in order to minimize waste without sacrificing high quality data.  This ultimately proves that science including high-throughput screening specifically can evolve to incorporate environmentally friendly techniques while continuously advancing the field.

    Carleen Klumpp-Thomas

    NIH/NCATS

    Carleen Klumpp-Thomas currently leads the Automation Group at the National Center for Advancing Translational Sciences (NIH/NCATS) in Rockville MD. Carleen manages and runs all of the automated screening platforms for NCATS Research Services Section (RSS). RSS’ multi-disciplinary capabilities enables the ongoing operation of all of NCATS’ research activities. These automated platforms perform a wide variety of experiment types ranging from biochemical, cell based, RNAi and other existing and novel assay technologies. Carleen’s automation and engineering expertise has been critical for projects ranging from cancer to Ebola to Parkinson’s disease.  The advanced instrumentation, protocols and methods are necessary to keep NCATS at the leading edge of scientific research and Carleen manages these requirements with ease, all while staying in constant communication with all researchers. Carleen earned her B.S. degree in Bioengineering from Syracuse University and her M.S. in Biomedical Engineering from NYU Tandon School of Engineering.

  • An Acoustic Microfluidic Device for Hematopoietic Stem Cell Enrichment from Whole Blood

    Contains 1 Component(s) Recorded On: 02/06/2018

    Here, we present a system that continuously separates HSCs from both healthy and diseased whole blood using acoustically-mediated separation in a plastic microfluidic device. We and others have previously demonstrated acoustic separation of bacteria, beads, and liposomes from blood cells, but this is the first report showing enrichment of HSCs directly from patient samples.

    Emerging cell therapies require efficient methods for purification of target cells prior to subsequent processing. In the case of stem cell-based therapies large numbers of hematopoietic stem cells (HSCs) must be collected and purified from patient peripheral blood; a challenging task because even after mobilization, the concentration of HSCs in the collected product is typically less than 1% of all cells. Existing processing techniques, such as density gradient centrifugation and subsequent magnetic separation, achieve some of the requirements, however, they often provide low yield, are costly, time-consuming, and labor intensive. Acoustic separation has emerged as a versatile technology for flow-through liquid handling and particle manipulation. The technique relies upon differences in the size, density, and compressibility of various blood components in order to achieve rapid label-free discrimination between target and off-target cells.

    Here, we present a system that continuously separates HSCs from both healthy and diseased whole blood using acoustically-mediated separation in a plastic microfluidic device. We and others have previously demonstrated acoustic separation of bacteria, beads, and liposomes from blood cells, but this is the first report showing enrichment of HSCs directly from patient samples. In addition, because our microchannel is constructed entirely of polystyrene, it is suitable for scale-up to clinically relevant processing rates, with the potential for flow rates approaching 100 ml/hr. Our device consists of a microchannel mounted on a piezoelectric actuator and a temperature-controlled stage. The actuator excites an acoustic standing wave within the fluid cavity, transverse to the flow direction. This standing wave exerts a force on blood cells which drives them toward the centerline of the flow. Larger and denser cells experience a larger force compared to smaller and less dense cells, and are more strongly focused. At the downstream end of the channel, a trifurcating outlet allows for the separation of strongly focused cells (e.g., red blood cells and neutrophils) from those that are weakly focused  (e.g., HSCs and lymphocytes). In this work the system is tuned to enable the separation and collection of HSCs.

    We achieve enrichment of the HSC population (CD34+ as % total white blood cells) from 9% to 22%, a factor of 2.4x, starting from unpurified whole patient blood. This enrichment was achieved in a single pass through the device with HSC recovery of 40% and reduction of the red blood cell concentration by 62%. These figures can be improved by multiple passes through the system and by device optimization. Our results demonstrate that we are able to efficiently and specifically purify HSCs from whole blood in a continuous flow-through device. In addition, our device is fabricated from low-cost components and is straightforward to operate, giving it the potential for future use in sample purification for cell therapy.

    Charles Lissandrello

    Draper

    Charles Lissandrello is a Senior Member of the Technical Staff in the Nano Structured Materials group at Draper. He received his Ph.D. in Mechanical Engineering at Boston University in 2015, where he conducted research with Kamil Ekinci on the transition from hydrodynamic to kinetic gas behavior in fluid systems far from equilibrium. At Draper, he has worked with a team of collaborators to develop novel microfluidic devices for biological sample processing.

  • DNA Encoded Library Selection Method to Rank Order Primary Hits by Affinity

    Contains 1 Component(s) Recorded On: 02/06/2018

    DNA encoded libraries are now routinely employed as part of reductionist lead generation campaigns in Pharma. The large number of compounds contained in many of these libraries (> 1 Billion) when combined with modest hit rates (0.1%) often result in thousands of potential hits.

    DNA encoded libraries are now routinely employed as part of reductionist lead generation campaigns in Pharma. The large number of compounds contained in many of these libraries (> 1 Billion) when combined with modest hit rates (0.1%) often result in thousands of potential hits. The compounds are generated as large combinatorial mixtures and “selected” for affinity to the target of interest. As a result the first step in hit triage is to resynthesize the compounds of interest without the DNA tag and confirm that the observed affinity for the target translates into the desired functional activity. Here we present experimental protocols and informatics methods that can estimate the affinity of the hits in the DNA encoded library mixture, thus enabling the incorporation of a ligand efficiency estimate into the decision making process for compound resynthesis. 

    Jeff Messer

    GlaxoSmithKline

    Biology, Drug Discovery, Informatics, Scientific Computing, Software Engineering

  • An end-to-end automated solution for provisioning compounds from a large liquid library for target and hit identification efforts

    Contains 1 Component(s) Recorded On: 02/06/2018

    Pfizer, like many large pharma, holds millions of compounds in its collection. This presentation will detail the migration from a single-use tube technology requiring a fit-for-purpose building, to a standard lab footprint automated system utilizing multi-use containers and acoustic-based liquid dispensing.

    Pfizer, like many large pharma, holds millions of compounds in its collection.  This presentation will detail the migration from a single-use tube technology requiring a fit-for-purpose building, to a standard lab footprint automated system utilizing multi-use containers and acoustic-based liquid dispensing.  The resulting solution is seamlessly integrated with a commercially available Enterprise compound-to-assay requesting tool.  Any member of the global organization has the ability to order compounds for plating to any number of assays, with plate shipment to any location. The solution, called Hit ID Provisioning System (HIPS) incorporates rule-based automation behavior in making final deliverables of assay ready plates that ensure plate quality under minimized stock consumption.  A novel compound binning algorithm compensated for the limitations of current acoustic liquid handling logistics.  Key considerations around implementation of new technology platforms will be reviewed in evaluating how the HIPS was rolled out to enable Pfizer researchers and collaborators access to the compound collection without interruption while improving plate quality and saving resources.

    Keith Miller

    Pfizer WR&D

    Keith obtained his Bachelors in Biomedical Engineering from Cornell University and an MBA from the University of Connecticut.  He has been working in Pharma at Pfizer since 2000, and is currently located at the Groton, CT campus.  Keith heads up the Compound Management & Distribution group's hit identification lab.  His group oversees Pfizer's largest compound collection - roughly 4 million unique compounds stored in liquid format.  Keith's lab serves not only as the stewards for this collection, but also oversees the plating & distribution of compounds to support Pfizer's global portfolio of plate-based early discovery projects.  Prior to his current role, Keith has worked with a broad range of research disciplines, either directly or through his expertise with automation and liquid-handling instrumentation: High-Throughput Screening, Analytical Chemistry, Protein Crystallization, NMR Screening, Protein Cloning & Expression, and Biophysics. 

  • Protein quality and assay development for successful DNA-encoded library screening

    Contains 1 Component(s) Recorded On: 02/06/2018

    In this presentation, case studies of protein target assessment enabling DNA-encoded library screening success will be shown.

    Active hit identification from DNA-encoded library screens is driven by high quality protein targets. Because the effective concentration of individual DNA-encoded library molecules in the screen is very low, the immobilized protein target concentration must exceed the dissociation constant to drive protein-library molecule binding. The protein target should also be a consistent conformation and without aggregates so the resulting data is associated with a single protein form. Ensuring that the immobilized protein target maintains biochemical or biophysical activity in the course of selection biases the outcome to functionally active hits. In this presentation, case studies of protein target assessment enabling DNA-encoded library screening success will be shown. 

    Allison Olszewski

    X-Chem

    Dr. Olszewski joined X-Chem in 2016 as Associate Director of Protein Biochemistry. She developed Avimer panning techniques against membrane-bound targets at Avidia, Inc. (acquired by Amgen in 2006) from 2006 to 2007. From 2007 to 2015 she worked at GlaxoSmithKline, where she expanded the use of DNA-encoded library technology to non-soluble targets. Before joining X-Chem, Dr. Olszewski managed the screening hit validation effort, and developed protein-protein interaction assays for the Small Molecule Assisted Receptor Targeting (SMART) technology platform at Warp Drive Bio. Dr. Olszewski received her B.S. in Biochemistry from the University of Delaware, and her Ph.D. in Organic Chemistry/Chemical Biology from the University of California, Irvine.

  • A high-throughput arrayed CRISPR/Cas9 functional genomics approach to study NF-kB signaling

    Contains 1 Component(s) Recorded On: 02/06/2018

    We undertook an array-based CRISPR/Cas9 screen at 800 kinome-scale to investigate mediators of TNFα-mediated NF-κB activation. Our results demonstrate the potential for genome-scale screens at high specificity using CRISPR/Cas9 and in a wide variety of cell backgrounds and phenotypes.

    CRISPR/Cas9 is increasingly being used as a tool to prosecute functional genomic screens.  Array-based CRISPR/Cas9 screens offer the ability to interrogate more diverse phenotypes than pool-based screens but their execution at scale in difficult to transfect cells brings challenges.  We undertook an array-based CRISPR/Cas9 screen at 800 kinome-scale to investigate mediators of TNFα-mediated NF-κB activation.  We used an ME180 cell line stably expressing Cas9 and a beta-lactamase reporter of Nf-κB activation alongside a developed lentiviral sgRNA library.  Hits were validated by confirmation of DNA insertion/deletion and screening orthogonal reagents.  Screening data quality was within acceptable limits (Z’>0.6) and genes associated with canonical NF-κB signalling were identified.  Our data provide unique insights into approaches and tools to explore novel biology with array-based gene editing in cellular assays.  Our results demonstrate the potential for genome-scale screens at high specificity using CRISPR/Cas9 and in a wide variety of cell backgrounds and phenotypes.

    Patrick O'Shea

    Astrazeneca

    Patrick O'Shea is a Senior Scientist in the High Content Biology group within Discovery Sciences at AstraZeneca in Cambridge UK; a multidisciplinary R&D unit with a global remit. The team develops novel phenotypic assays and uses high-content and functional genomic approaches to understand novel biology, provide an early insight into compound toxicity, and to characterize compound mechanism of action in disease relevant models. Prior to joining AstraZeneca, Patrick studied Pharmacology at the University of Leeds. He completed a PhD at Imperial College London, studying the developmental effects of thyroid hormones in bone and cartilage, and was a postdoctoral fellow at Imperial College London and at the National Cancer Institute, NIH. 

  • Genome-wide CRISPR-mediated Gene Disruption Presents a Shortcut to Acquired Resistance that Reveals Small Molecule Mechanism of Action

    Contains 1 Component(s) Recorded On: 02/06/2018

    Phenotypic screening in small molecule drug discovery presents the opportunity to discover novel therapies, but thorough identification of a small molecule target remains an obstacle. To address this challenge we applied whole-genome pooled CRISPR screening as a Shortcut To Acquired Resistance in Search of mechanism (STAR-Search).

    Phenotypic screening in small molecule drug discovery presents the opportunity to discover novel therapies, but thorough identification of a small molecule target remains an obstacle. To address this challenge we applied whole-genome pooled CRISPR screening as a Shortcut To Acquired Resistance in Search of mechanism (STAR-Search). This strategy uses CRISPR to generate a population where individual cells each possess a distinct targeted mutation. This comprehensive pool of mutations is then subjected to positive selection, which enriches cells that acquire resistance to compound treatment. The resistance is caused by targeted mutations that are readily identified by sequencing the stably integrated targeting construct. We hypothesize that the identity of gene disruptions underlying resistance can reveal mechanism of action or factors proximal to the direct target. Our group has successfully applied STAR-Search to multiple phenotypic screening hits, thus demonstrating its strong potential as a tool in target identification/validation. Our application of STAR-Search examined three small molecules that each elicits cytotoxic effects against a unique spectrum of cancer lines. CGS-18, which preferentially induces apoptosis in breast cancer lines, was dosed onto MDA-MB-468 cells stably transduced with the Brunello CRISPR gRNA library. Cells that survived CGS-18 selection showed enrichment of gRNAs targeting a single gene SULT1A1. MDA-MB-468 cells also undergo apoptosis in response to CGS-59 treatment, so this positive selection was performed in parallel with the previous screen. In this selected population, gRNAs targeting MGST1 were the most highly enriched. Validation experiments have confirmed that individual disruption of SULT1A1 or MGST1 confers resistance to CGS-18 or CGS-59, respectively. The third small molecule, CGS-85, displayed selective killing of multiple myeloma cell lines. This compound was profiled in the BioMap Diversity+ Panel where its phenotypic effects showed strong correlation to the reference database profile generated by the oxidative phosphorylation inhibitor oligomycin. LP-1 cells transduced with the CRISPR library that survived either CGS-85 or oligomycin selection showed enrichment of gRNAs targeting a large number of genes, but this group converged on a common mechanism: mitochondrial oxidative phosphorylation. Despite substantial overlap between the majority of screening hits, prominent differences suggested distinct direct molecular targets. Subsequent enzyme assays showed CGS-85 potently inhibits isolated mitochondrial complex I, whereas oligomycin confirmed as an inhibitor of complex V. Together these examples illustrate the potential of STAR-Search to reveal small molecule mechanisms of action and specifically uncover novel biological connections due to the comprehensive and systematic nature of the genome-wide CRISPR targeted disruptions.

    Jon Oyer

    Abbvie

    Jon Oyer works within the Target Identification & Validation group of the Genomic Research Center at AbbVie. This group specializes in applying a diverse stack of technologies and methods to the challenge of small molecule characterization. A partial list of these collaborative efforts include transcriptome analysis, proteomics, phage display, cellular thermal shift assays, and functional genomic approaches. Jon received his undergraduate degree from University of Washington before completing his graduate studies in Molecular & Medical Genetics at Oregon Health & Science University. Prior to joining AbbVie, Jon also conducted postdoctoral research studying epigenetic regulation in embryonic stem cells with Gail Mandel at Oregon Health & Science University and epigenetic disruptions that drive hematological malignancies with Jonathan Licht at Northwestern University.