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SCREENING TECHNOLOGY
  • Screening Facility
  • Computers, Servers, Network and Data Backup Systems
  • LIMS and HTS/HCS Analysis Software
  • Screening Workflow
  • High Throughput Screening
  • High Content Screening
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    Screening Technology
     
    bullet point  Screening Facility
     
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    The PMLSC Screening Facility, located on the ninth and tenth floors in the Biomedical Science Tower 3, provides investigators with the ability to run High Throughput and High Content Screens. The following equipment and resources are available for investigators:

  • Automated compound storage and retrieval system.
  • Liquid handling robots for compound transfer, compound cherry picking and filling assay plates.
  • Multimode plate readers with luminescence, fluorescence, fluorescence polarization, time-resolved fluorescence, and absorbance detection capability.
  • High Content imaging screening robots with the live cell and kinetic screening options.
  • A cheminformatics Oracle database and an High Content screening image storage and analysis database.
  • Screening microscopes that image wells from 96-, 384- and 1536-well assay plates.
  • Tissue culture hoods and incubators.
  • Multichannel pipettes.
  • Plate Sealing robot.
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    bullet point  Computers, Servers, Network and Data Backup Systems
     
     
    The PMLSC has:
  • (10) Dell Precision Dual Core workstations
  • (77) PCs
  • (3) Mac computers
  • 9 servers: (2) Dell PE 1850’s Domain controller servers; (1) Dell PE 2850 Windows file server with 8 GB memory and two Dual Core 2.8GHz/2MBx2 CPUs; (1) Dell PE 2850 Oracle Database and IDBS ActivityBase LIMS server with 8 GB memory and two Dual Core 2.8GHz/2MBx2 CPUs; (1) Dell PE2950 server with 4GB memory and two Dual-Core 2.0 GHz/1333 MHz FSB/4MB Cache CPUs attached to a 6TB SATA disk storage to house the a ArrayScan® IV Cellomics Store database; (1) Dell PE2950 server with 16 GB memory and two Quad Core 66GHz/1333MHz FSB/4MBx2 Cache CPUs with 16TB SAN storage to house the ArrayScan® VTI Cellomics Store database; (1) high-performance Dell 2950 application server with four analysis engines and two high-speed quad-core CPUs to house the Definiens image intelligence suite; (2) Dell PE2850 server with 4 GB memory and two Dual Core 2.8GHz/2MBx2 CPUs to house one PubChem raw data FTP/WEB server to provide the public access to the raw data and images from MLSCN screening campaigns completed by PMLSC.
  • A scalable EMC CX300 Storage Area Network (SAN) solution has been deployed and upgraded to be the central data warehouse with 50 TB of formatted storage configured with 7 TB of high performance fiber channel disk storage, and 43 TB of SATA disk storage. The SAN is connected to a Dell PowerVault™PV-TL4000 and PV-124T tape drives with a 64 tape slots for data backup and recovery.

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    bullet point  LIMS and HTS/HCS Analysis Software
     
     
    The PMLSC has (3) IDBS ActivityBase LIMS Biology seats, (3) IDBS XE Seats (3) IDBS XLfit seats, (2) IDBS ActivityBase Chemistry seats, (1) IDBS Protocol Transfer Assistant, (7) IDBS SARview seats, (10) Spotfire® data visualization licences, (3) Leadscope Enterprise 2.4. 15-6 Chemical Structure analysis software seats. Cellomics platinum package of Bio-Application image analysis algorithms (V2 & V3) for their ArrayScan® imaging platforms; Cell Cycle, Cell Health Profiling, Cell Motility, Cell Spreading, Compartmental Analysis AS, Cyto-Cell Membrane, Cyto-Nuc Translocation, Extended Neurite OG, GPCR Signaling, Micronucleus, Molecular Translocation, Morphology Explorer, MP Cytotoxicity, Neuronal Profiling, Neurite Outgrowth, Spot Detector, Target Activation, and Tube Formation. (3) vHCS Discovery Toolbox and (3) vHCS View licenses. Molecular Devices ImageXpress application and informatics software package with (10) MetaXpress and AcuityXpress licenses and (1) parallel computation Cephalopod package with 16 Cephalopod agent license seats. Definiens image intelligence suite for custom image analysis including; (1) developer, (1) architect, and (1) Cellenger license with (4) data analysis engines.
     
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    bullet point  Screening Workflow
     
    Click to view large Screening System Architecture.JPG   
     

    At PMLSC, the LIMS (Laboratory Information Management System) software is being deployed and implemented to manage the work flow among the compound storage and retrieval, compound transfer, compound cherry picking, assay design, screening detection, screening quality control, hit detection, SAR report and PubChem screening data publication.

    Also the Electronic Laboratory Notebook will be deployed to provide the scientists with a single access point for capturing experimental data and performing scientific tasks.


     
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    bullet point  High Throughput Screening
     
     

    High throughput screening has become the dominant tool in the drug discovery process. In its earliest form, drug screening typically involved antimicrobial formats, however rapid progress in the fields of genomics, proteomics and molecular biology has expanded the target landscape beyond microbial targets, to both increase the numbers of potential drug targets, and facilitate development of assays to screen these targets. In parallel with these changes, developments in robotics and combinatorial chemical synthesis have driven the production of very large numbers of compounds with potential for pharmacological activity. The need to screen large libraries of chemical compounds against multiple targets has stimulated improvements in assay technology, instrumentation, and automation that evolved into the field of HTS and has revolutionized the field of drug discovery. In recent years, advances in miniaturization, parallel processing and data management have enhanced efficiency by controlling costs and increasing throughput thereby supporting the evolution of multiple platforms for lead generation.

    Upon selection of a target, a lead generation strategy is defined which typically includes HTS as the first step – the “primary” assay, followed by hit confirmation – “secondary” assays, which precede lead validation and optimization. Many factors can influence the format of the assays employed and their positioning in the screening paradigm; the type of pharmacological information sought, the target class, throughput, cost, and other logistical or practical considerations. Cell based screens and biochemical or isolated target screens each have their merits. Biochemical assays which utilize isolated enzymes, proteins or receptor preparations are generally amenable to homogeneous assay formats and miniaturization, potentially identify the majority of chemotypes that interact with the target, and can provide target specificity information. In contrast, issues like cell membrane permeability and compound cytotoxicity may limit the diversity of chemotypes and pharmacophoric information obtained from cell based screens, and cellular assays are more challenging to miniaturize or automate.

    The purpose of HTS is the interrogation of large chemical collections in the context of a biological target to accurately identify active chemotypes. To achieve this purpose, screens must be configured to provide a robust, reproducible signal with adequate throughput to screen large compound libraries. Since the activity or inactivity of any given compound in an HTS will typically be determined in a single well at one concentration, the assay signal window (dynamic range) must be sufficiently rugged to provide adequate separation between the maximum and minimum responses, and should enable the response to active compounds to be discriminated from the background variability (noise) associated with the top and bottom of the signal window. In addition to studies designed to validate the kinetics and pharmacology of the assay, efforts are made to optimize the signal window and/or variability of the assay in the context of a number of variables dictated by the automated process; DMSO tolerance, reagent stability, and signal stability. Superimposed upon assay development parameters associated with biochemical HTS formats, the implementation of cell-based screens present additional challenges; generation and/or characterization of an appropriate cell model, production of sufficient cells for HTS, plating cells for the assay, effects of compound exposure, and capture of the assay signal.



     
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    bullet point  High Content Screening
     
     

    High Content Screening (HCS) is an extension of a revolution in cell analysis that began about 3 decades ago when fluorescence labeling technologies were combined with electronic imaging technologies and used to study individual cells on a light microscope. The innovation that drove the development of HCS, and what distinguishes HCS systems from the many confocal and wide field microscopes, is the integration and automation of the entire analytical process. The first HCS platforms were introduced to the drug discovery market by Cellomics, Inc. (Pittsburgh, PA) starting in 1997. There are now more than ten models of HCS imagers established in the market. HCS platforms automate the capture and analysis of fluorescent images of millions of individual cells in tens of thousands of samples on a daily basis, and have made fluorescence microscopy and image analysis compatible with the needs of drug discovery and systems cell biology. Through selection of appropriate probes, antibodies, fluorescent protein fusion partners, biosensors, environmentally sensitive probes and stains, fluorescence microscopy can be applied to many drug target classes, may be configured for simultaneous multiple target readouts (multiplexing), and can provide information on distributions and cell morphology in addition to simple intensities. Image based assays therefore provide multi-parameter quantitative and qualitative information beyond the single parameter target data typical of most other assay formats, and thus are referred to as high “content” assays. In recent years there has been a growing trend in drug discovery towards the implementation of cell based assays where the target is screened in a more physiological context than in biochemical assays of isolated targets. Fluorescence microscopy, whether confocal or wide field, is one of the most powerful tools that cell biologists can use to interrogate bio-molecules and investigate the molecular mechanisms of the cell. Automated imaging platforms are therefore being deployed throughout the drug discovery process; target identification/target validation, primary screening and lead generation, hit characterization, lead optimization, toxicology, bio-marker development and diagnostic histopathology. Furthermore, these platforms are now spreading into the research markets for application in high throughput biology.

     
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