Neil Carragher: Drug Discovery
Cellular Pharmacology and Phenotypic Profiling
Established in 2011 under the leadership of a former industry expert in cell-based screening across various disease areas, the Edinburgh Cancer Development Unit (ECDU) implements industry standard, robust, quantitative, high specification assays and screens in an academic environment.
The core team specialises in cancer research based on a suite of high content, high throughput imaging technologies. Our latest development has been the establishment of the Edinburgh Phenotypic Assay Centre (EPAC) at the Queen's Medical Research Institute (QMRI) to expand these activities across disease areas. At the EPAC the focus is on the development of novel, disease-relevant, phenotypic screening assays across multiple disease areas.
High Content Imaging
The IncuCyte ZOOM is a compact dual fluoresecent and brightfield imaging microscope that can be placed within a standard tissue culture incubator. IncuCyte software enables remote scheduling of sequential image acquisitions and dedicated kinetic image analysis software further facilitates the longitudinal analysis of cell morphology and phenotypic markers within optimal environmental conditions (Isherwood, B.et al.,. 2011. Live cell in vitro and in vivo imaging applications: accelerating drug discovery. Pharmaceutics 3, 141-170.). The IncuCyte ZOOM is currently situated within a standard Heracell 240i incubator suitable for monitoring live cell phenotypes under normoxic or hypoxic conditions. The ECDU IncuCyte ZOOM possesses a suite of image analysis application modules and ECDU researchers are currently engaged in the development of novel 2D and 3D in vitro assays suitable for live cell longitudinal analysis of specific cancer phenotypes.
The ImageXpress MicroXl platform is a high content instrument from Molecular Devices. Incorporation of a large sensor scientific CMOS camera together with LED solid light source provides enhanced optical sensitivity and image quality over standard high content systems. New MetaXpressTM software solutions such as a “Digital Confocal Option” and “Custom Module Editor” provides increased capability and flexibility to customize image analysis routines for quantification of defined phenotypes. The AcuityXpressTM software facilitates quality control assessment across multiple plates and tissue slides and incorporates multivariate statistical and similarity profiling tools to exploit multiparametric phenotypic data. The ImageXpress platform represents a fully equipped high content solution integrated with plate handling robotics (PAA Scara 4 robot), barcode reader and an extensive image-informatics suite (MetaXpressTM and AcuityXpressTM software) that stream-lines; complex high-content analysis routines; data analysis; image storage and review.
In addition to using proprietary software solutions (including close collaboration with high content instrument and software suppliers) we have also established a number of ongoing collaborations with the academic high content imaging community to develop and apply novel image analysis and informatics solutions to advance high content phenotypic profiling. We are currently collaborating with Guido Sanguinetti (School of Informatics Edinburgh) and Anne Carpenter, Director of the imaging platform at the Broad institute of Harvard and MIT to apply opensource image analysis solutions and novel machine learning methods and statistical models to quantify subtle cellular phenotypes and classify distinct cell subpopulations from mixed cultures and drug-induced phenotypes from multi-parametric image based datasets. These novel approaches have demonstrated utility in quantifying both pre-defined and un-anticipated morphological cell phenotypes following drug screening in cancer cells.
We are also founding members of the European Cell based Assay Interest group (EuCAI) consortium which represents a network of the leading European academic centres with extensive experience in high content imaging, image analysis and machine learning across multiple disease areas. The objectives of EuCAI are to share best practice in high-content imaging and advanced cell based assays and develop the multidisciplinary skill sets required to fully exploit high content analysis through collaboration and training networks.
In order to facilitate our drug discovery research we have invested in several pieces of liquid handling equipment. These include two Biomek FX robots with attached Cytomat, primarily used for compound dilution, plate reformatting and assay set up, as well as an EpMotion 5070 housed in a Class II biological safety cabinet which is available for setting up long term assays and level II biohazard work.
Compound Libraries for High Throughput Screening
In the process of furthering our drug discovery research we have procured libraries of annotated reference compounds and FDA/EMEA approved drugs as well as novel compounds. Our compound sets are stored and managed at Bioascent Discoveries Ltd facilities in Newhouse, Biocity-Scotland UK which was selected as the centre of excellence for compound management of the European Lead Factory compound collection. As part of the compound management process, our compounds are delivered by Bioascent as single use, assay-ready plates.
Our 12,000 compound diversity set represents the diversity of a 120,000 compound collection. This compound library was designed by medicinal chemists within the pharmaceutical industry (formerly Merck). Compounds were selected for drug-like properties and suitability for cell-based screening and as such the library represents an excellent starting point for phenotypic screening.
Our annotated reference library is composed of the Screen-Well kinase, protease and epigenetic inhibitor libraries (175 compounds) from ENZO and 120 manually-curated compounds representing a broad range of mechanism-of-action classes.
Our Prestwick FDA/EMEA Approved Drug Library represents 1,280 compounds that will support repositioning of existing drugs already in the clinic to patients, if suitably potent hits are discovered.
Reverse Phase Protein Microarrays (RPPA)
RPPA represents a highly sensitive and quantitative high-throughput antibody-based proteomics methodology. Key applications of our RPPA platform include: lead/candidate drug mechanism-of-action profiling; quantifying on and off-target activities at the post-translational level and protein biomarker discovery in clinical and preclinical studies linked to dose/phenotypic outcome/context. The Aushon 2470 and ZeptoMARK RPPA platforms installed within the ECDU incorporate the latest technical advances in RPPA integrated with near-infrared detection technology to provide industry leading sensitivity and precise quantification of changes in protein levels and post-translational modification. Together with Zeptosens-Bayer we have validated a portfolio of over 360 antibodies that can be used to simultaneously profile multiple pathway signaling response states (including multiple canonical, non-canonical and key cancer driver pathways) across precise time- and dose-studies. Our participation in leading a global RPPA society ensures we remain at the cutting edge of RPPA technology and best practices.