DPFS Resource request (University of Sheffield) A High-content high-throughput Screening Facility in an Academic Setting

Lead Research Organisation: University of Sheffield
Department Name: Medicine and Biomedical Science

Abstract

Thanks to the recent multi-national efforts to sequence the entire genomes of species as diverse as worms and humans, we now know that humans and lower organisms share remarkably similar sets of genes. This finding has transformed our understanding of human biology, through the realisation that many of the cellular and biochemical processes controlled by these genes in simple organisms also operate in ourselves. Most of our knowledge about the control of cells and organ systems has come from the study of a few so-called ?model organisms?, especially the fruit fly Drosophila melanogaster, and the tropical fish Danio rerio (commonly known as the zebrafish). Studies of such organisms have the potential to revolutionise our understanding, diagnosis and treatment of human disease, a premise that underpins the activities of the MRC Centre for Developmental and Biomedical Genetics. A key aim of the Centre is to encourage the rapid translation of our basic research findings into clinical application. This proposal will facilitate such translation by providing the means to screen for small molecules that can modulate pathogenic processes in model organisms. Such small molecules will provide powerful tools for probing the molecular basis of these processes and in some cases will provide leads for the developments of new drugs for their treatment.

Technical Summary

The MRC Centre for Developmental and Biomedical Genetics (CDBG) brings together basic and clinician scientists using non-mammalian model organisms to understand the genetic basis of human disease, knowledge that will be essential for the development of novel therapies. In recent years, several groups within the CDBG have established disease models that provide a basis for high content screens. The primary objective of this proposal is to establish a facility for, high-throughput screening of small molecule libraries using these high content biological assays with the dual aim of identifying novel reagents for continued research and the establishment of systems to speed the identification and validation of lead compounds for drug discovery. In contrast to conventional drug screening programs where efforts are focused on a relatively small number of disease-relevant target pathways and proteins, the whole organismal nature of our models provides the potential for identification and validation of reagents affecting multiple pathways in the pathogenic process. A second application of the facility will take advantage of more conventional tissue culture based assays that are currently being devised for use in RNAi screens. The shared Screening Facility will have the flexibility to accommodate many different types of assay. Individual researchers will carry out the vast majority of work for their own screening projects (see CDBG component outline applications), but the Facility will provide the capacity for integrated management and automation of assay protocols and for access to business and pharmaceutical pipelines. Further, the Facility will enable and underpin future translational needs that lie at the heart of the University?s strategic vision: as functional genomic/proteomic datasets of patient-based materials accumulate in the Academic Medical Centre, these will integrate with the information from high throughput screens from cell-based and animal model systems in the CDBG. The Facility will therefore support a bidirectional flow of information, integrating mutually informative data from basic and clinical platforms.

Publications

10 25 50
 
Description BBSRC Excellence with Impact Competition
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Influenced research clinician training
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Participation in PERA organised JISC 'open innovation' discussion group
Geographic Reach National 
Policy Influence Type Participation in a national consultation
 
Description Royal Society Summer Exhibition
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
 
Description FP7-Health (FVE)
Amount £306,000 (GBP)
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 07/2010 
End 12/2015
 
Description UoS Knowledge Transfer Scheme (SR)
Amount £9,696 (GBP)
Organisation University of Sheffield 
Sector Academic/University
Country United Kingdom
Start 01/2009 
End 12/2009
 
Description WT RNAi Screening Centre (MZ/AW)
Amount £916,912 (GBP)
Organisation Multiple Myeloma Research Foundation 
Sector Charity/Non Profit
Country United States
Start 11/2008 
End 10/2013
 
Title A data repository for high throughput screening data (MM) 
Description The system developed is an end-to-end collection, processing and presentation system to support the other sections of this grant. The Data Organisation System (DOS) accepts raw images as produced by optical scanning equipment (primarily microscopes purchased through this award) as input and using an agreed code embedded into the filename or external metadata file. This award inspired the inception of a high performance data storage system in a collaborate effort between researchers from the MRC- CDBG, the Welcome Trust funded RNAi Facility and the Light Microscope Facility. The award used this hardware to implement an advanced file archiving / indexing system based on file fingerprinting such that only links to files are stored in the database and the files are stored on disk. Should the original files move then relinking them correctly in the database is a trivial matter. In the prototype system researchers can view loaded images and assess the images as to whether the result is considered a 'Hit' or whether the compound was considered toxic. The structure of the database has been designed such that all results are attached to a central unique combination of Screen-Plate-compound for library-Well. This allows the database to record more complex quantitative parameters (eg. Fluorescence intensities, absence of fish features, links to external analyses). Details of the compound libraries used for screening are loaded into the database with annotation pertinent to it stored for easy retrieval. A guiding principle of the database is that all the data either loaded in or collected by the interfaces (WWW, SQL access) is stored in flat files as far as is possible and that the entire database can be rebuilt from this using computer code. This allows ease of data integration with other external analysis packages and data entry using familiar spreadsheet interfaces. Template Spreadsheets have been constructed for this purpose that include rudimentary data validation. Further different screens can then be linked together to form 'Investigations' such as primary screen, confirmation of Hits in a smaller replicate screen, a follow-up secondary screen and similar alternative results found in literature searches. Software has been developed based on practical experience gained in the laboratory to identify problems in the library annotation (e.g. multiple compounds listed in the same well, duplicate compounds present, consistency checking of library rearrangements) and present these for further investigation / correction. The chemical structures of compounds in the library are being compared to both each other in the same library, to those in other libraries and publicly databases to identify similar compounds, allow decisions about the overlap in libraries and to improve annotation respectively. The analysis continues but there are clearly patterns within the comparison even when simple atoms connectivity is considered using Open Source Cheminformatics packages (OpenBabel) implementing classical distance measures. Through a collaboration at Erasmus MC (Rotterdam, The Netherlands) an optimally ordered heatmap using specialist software they have access to group compounds together into meaningful clusters has been prepared in preliminary form. This is useful for giving insight to non-domain specialists as to the relationships between compounds contained in libraries. 
Type Of Material Biological samples 
Year Produced 2009 
Provided To Others? Yes  
Impact Support of scientific screens internally. 
 
Title Establishment of a drug screening facility (MP) 
Description The drug screening facility is now able to perform all stages of the drug screening pipeline. We have assembled equipment for compound handling and embryo production through to multiple assay designs and hit detection, using a combination of automated and manual methods. In particular we have collaborated with Ash Biotech to increase throughput of embryos into assay plates and hit detection of fluorescent-based assays. We have increased embryo production using a miniMEPS tank (aquatichabitats), increased throughput for the in situ hybridization screen with an in situ hybridization machine (Intavis) and increased hit detection with a customized microscope with a motorized stage from Nikon that is specifically for multiwell plates and can be used for brightfield and fluorescent images. We can screen for behavioural changes using the Zebralab system from Viewpoint 
Type Of Material Improvements to research infrastructure 
Year Produced 2011 
Provided To Others? Yes  
Impact Seventeen assays have been completed using the facility and 10 others are in the pipeline. 
 
Title The development of a new assay protocol for screening the effects of small molecules on zebrafish embryos by in situ hybridization (SB) 
Description This protocol uses mesh bottomed plates (Millipore) throughout; from the addition of the drug, through the in situ hybridization machine (Intavis) and the image capture on a customized Nikon microscope. It has a number of advantages over existing methods including increased throughput, reduction in transferring of embryos to reduce the damage to the embryos and sample mixing errors, and high quality automated image capture for hit detection. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact We have screened 8,000 compounds using this method. 
 
Title Zebrafish epilepsy model (VC) 
Description A novel model of epileptic seizure induction in zebrafish and a high throughput in situ hybridisation-based assay for seizure-resolving compounds have been developed. This model and the assay have been exploited to identify novel small molecules with anti-convulsant activity and may therefore have potential as anti-epileptic drugs. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact Selected Oral Presentation at EUFishBiomed COST-ACTION Workshop on Zebrafish Neurophysiology an Behaviour. Queen Mary University of London 2010. HDAC inhibitor screen in zebrafish (Jan-June 2010). A collaboration with Karus Therapeutics funded by University of Sheffield Knowledge Transfer Project Fund. 
 
Description Customized microscope (SB) 
Organisation Nikon
Country Global 
Sector Private 
PI Contribution Collaborated to produce a customized microscope for automated imaging of bright field and fluorescent images in multiwall plates
Collaborator Contribution Collaborated to produce a customized microscope for automated imaging of bright field and fluorescent images in multiwall plates.
Impact NA
Start Year 2009
 
Description Development of new equipment to increase throughput of drug screening methods in zebrafish (SB) 
Organisation ASH Biotech
Country United Kingdom 
Sector Private 
PI Contribution Collaboration to produce new equipment for increasing throughput of drug screening methods in zebrafish. Over the past year we have contined to be involved in the product specification and design and testing of the equipment.
Collaborator Contribution Increasing throughput of drug screening methods in zebrafish. The collaboration resulted in two new pieces of equipment being developed. The Phenosight imaging system has been modified over the past year and the software is much improved. The embryo dispenser has also improved but is still in the beta version.
Impact Beta versions of two pieces of equipment are being evaluated. The Phenosight fluorescent plate reader that can take high speed images of transgenic zebrafish in multiwell plates and record the level of GFP in each embryo is now ready for marketing by ASH. This equipment is used extensively in the screening unit. An embryo dispenser that can accurately aliquot embryos into 96 well plates is being optimized. The beta version works well for 48 hpf embryos.
Start Year 2009
 
Description HDAC inhibitor screen in Zebrafish [Karus Therapeutics] (VC) 
Organisation Karus Therapeutics
Country United Kingdom 
Sector Private 
PI Contribution Characterisation of novel HDAC inhibitors as potential anti-epileptic drugs using molecularly focused screen in zebrafish.
Collaborator Contribution Characterisation of novel HDAC inhibitors as potential anti-epileptic drugs using molecularly focused screen in zebrafish.
Impact NA
Start Year 2009
 
Title Assay development 
Description Assay development zebrafish tools for translational research 
IP Reference  
Protection Protection not required
Year Protection Granted 2010
Licensed No
Impact Facilitated interactions with potential commercial partners. Awareness of the facility across the Academic Community: 1. Leading Edge Analysis Cell 139 p843-846 (2009) PMID: 19945369 [PubMed - in process] 2. HTS and hit finding in academia - from chemical genomics to drug discovery Drug Discovery Today 14 p1150-1158 (2009) Pages 1150-1158 Julie A. Frearson, Iain T. Collie
 
Title Identification of potential drug targets in Parksinson's Disease 
Description Identification of Potential drug targets in Parksinson's Disease. 
IP Reference  
Protection Protection not required
Year Protection Granted 2009
Licensed No
Impact None as yet.
 
Title Identification of potential drug targets for Parkinson's Disease 
Description Identification of potential drug targets for Parkinson's Disease 
Type Therapeutic Intervention - Drug
Current Stage Of Development Initial development
Year Development Stage Completed 2009
Development Status Actively seeking support
Impact none as yet 
 
Description Research translation 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Health professionals
Results and Impact Presentation to health professionals. On separate occasions, dissemination to potential industrial collaborators and consultants. Within the University, raising awareness of DPFS scheme.

Awareness
Year(s) Of Engagement Activity 2009
 
Description Royal Society Summer Science Exhibition - London 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact "Fishing for clues: why medical researchers are glowing with excitement". Members of the Centre for Developmental and Biomedical Genetics presented their research to school children, members of the public and invited Royal Society guests. We demonstrated our research using live zebrafish embryos (fluorescence microscopy), together with video and written presentations. We talked to the visitors and gave interviews.

Enthused and educated public in the area of our research. Request for an article in Catalyst magazine.
Year(s) Of Engagement Activity 2009