As such PDD is comparable to empirical screening, which was historically used in drug discovery before more target-based approaches became popular. Currently there is a resurgence in interest in PDD, driven by many factors, not least the limited success of target-based drug discovery.

One of the key features of current phenotypic approaches is the biological relevance of the assay systems deployed and in this respect the commercial availability of unlimited quantities of pure human cell types, particularly those derived from induced pluripotent stem cells, is having an impact.

Stem cells are fuelling the development of many new disease models and with high levels of translation to human biology and disease these phenotypic assays are increasingly being used in early toxicity testing. PPD has also been powered by recent advances of high content imaging systems, facilitating the rapid analysis of increasingly complex multi-parametric measurements of cellular phenotypes or biomarkers. PDD looks set to be with us for the foreseeable future, with a role alongside targetbased and other approaches to drug discovery.  

Phenotypic drug discovery (PDD) was extensively discussed in DDW last year1 and readers are strongly recommended to consult this excellent review for wider insight. However, for the purpose of this article it is worth beginning by restating a few definitions around which there is broad agreement. Phenotypic assays aim to quantity a phenotype or pathway in a physiological relevant system (typically a cell or whole organism) and make limited assumptions as to the molecular basis of how the system works. Phenotypic screening includes any screening where the molecular mechanism of action is not assumed and does not involve screening against an isolated target. In this context phenotypic screening is comparable to empirical screening or classical pharmacology which was historically used in drug discovery before more target-based approaches became popular. The current resurgence of interest and awareness of the value of phenotypic screening in drug discovery has been driven by a range of factors including: 1) limited success of target-based drug discovery, typically with the protein target in solution or over expressed in the cell; 2) the need to minimise the risk of late stage attrition due to poor efficacy or off-target activities; 3) the realisation that many of today’s first-in-class drugs with novel mechanisms of action came from phenotypic screening; 4) access to more relevant cells types (such as primary and stem cells), validated translational biomarkers and more predictive animal models of disease; and 5) advances of high content screening, facilitating the analysis of increasingly complex multi-parametric data. One of the key features of current phenotypic approaches is the biological relevance of the assay systems deployed and in this respect the commercial availability of unlimited quantities of pure human cell types, particularly those derived from induced pluripotent stem cells, is having a major impact. Phenotypic assays share a common feature, ie there must be high confidence in their translation to human biology and disease.

In August 2015, HTStec carried out an industrywide, global, web-based benchmarking survey on PDD. This survey set out understand and record the latest thinking around many aspects of PDD, including how it is being deployed, where phenotypic assays are most suited to be used, expectations of phenotypic screening, milestones achieved, future requirements for phenotypic primary screening and assay technologies and instrument platforms currently used in PDD. The results of the survey were publ ished in HTStec’s PPD Trends 2015 report2 and selected findings are now reported in this article together with vendor updates on technologies facilitating phenotypic screening assays.

Main features of PDD
Captures bio content (targets presented in a physiological context) was ranked the most important feature in understanding PDD. This was followed by closest to ‘in vivo’ as practical and then mechanism/ target agnostic. Ranked least important was mitigates target validation issues (Figure 1).

PDD assay types
The assay types that best fit within survey respondents’ definition/understanding of phenotypic screening assays were assays involving co-cultures of primary cells and assays involving primary cell cultures (both with 81% selecting). This was closely followed by 3D cell culture/2D co-culture assay models and assays involving multi-parameter high content imaging (both with 76% selecting). Assays involving beads in solution and ligand-binding assays least fitted well within respondents’ definition/ understanding of a phenotypic screening assay (Figure 2).

Main motivators for adopting PDD
Allows for discovery of unexpected biology was ranked the main motivator for wanting to adopt PDD. This was followed by mimetic of in vivo biology, system best represents disease pathology or biology and then enhances the discovery of innovative therapeutics and decreases ‘me too’ drug efforts. Least motivating was flat NME output of target-based drug discovery (Figure 3).

Main obstacles limiting adoption
Understanding the biological endpoints needed was rated the main obstacle limiting adoption of phenotypic screening assays. This was closely followed by target deconvolution, access to relevant cell models (eg primary human cells) and then choosing the best cell type. Rated least limiting was lack of appropriate chemical diversity (Figure 4).

Cell types most suited for phenotypic screening
Human primary cells were ranked as the cell type most suited (relevant) for phenotypic screening studies. This was followed by stem cells or iPSderived phenotypes and then primary cells of animal origin. Considered least relevant were native immortalised cells (Figure 5). Biological systems most used to investigate PDD Cell line monocultures were the biological systems currently most used by survey respondents to investigate PDD. This was followed by human primary cells; co-cultures/cell mixtures and then stem cells or iPS derived cultures. Least used were tissuelike models/xenografts (Figure 6).

Desired outcomes of phenotypic screening
Novel MOA which is differentiated from standard of care and to understand the functional responses were both equally rated as survey respondents’ most likely (desired) outcomes for phenotypic screening. They were followed by demonstrate in vivo activity and identify compounds selective in vitro. Rated least likely outcome was identify target of active compounds (Figure 7).

Usefulness of PPD
SAR observed in phenotypic assay system was the PDD project milestone most achieved to date without knowledge of the target. This was followed by cellular mechanisms have been identified, and then in vivo efficacy observed (Figure 8).

Phenotypic assays were ranked as most suited for the identification of novel therapeutically active molecules for drug discovery. This was closely followed by identification of novel molecular targets of therapeutic importance. Ranked least suitable was for si/sh RNA screening (Figure 9).

Agreement with statements about PDD
Survey respondents’ level of agreement with statements about PDD showed strongest agreement for ‘PDD is a solid trend that bridges the in vitro to in vivo gap, it is likely to progress in primary screening over the next few years’ and strongest disagreement for ‘PDD is being driven by upper management, not by the biology teams’ (Figure 10). 

Vendor updates of technologies useful in phenotypic drug discovery
In addition to screening compound libraries using phenotypic efficacy models, there is a benefit in qualifying hit and lead compounds with early-toxicity- indicating assays that are adequately quantitative, specific, reproducible and suitable for a variety of cell types. For example, the MitoTox™ assay range offered by Abcam (www.abcam.com/ mitotox) is a useful preclinical tool set with HTS compatibility to test for drug-induced mitochondrial toxicity. This is particularly useful for antibacterial and antiviral drug development given the prokaryotic origin of the organelle and the evolutionary similarity between its biosynthetic machinery involved in mitochondrial biogenesis and the bacterial/viral replication systems. More specifically, the MitoBiogenisis™ In-Cell ELISA has been developed in a 96-well format to screen for mitochondrial biogenesis as a standard component of early drug safety characterisation. The assay indicates drug-induced inhibition of both, mitochondrial transcription and translation by comparing mitochondrial DNA- and nuclear DNA-encoded protein levels in cultured or primary cells. Low mitochondrial biogenesis activity in response to drug exposure results in either increased free radical generation (reactive oxygen species, ROS), dysfunctional apoptosis or impaired energy metabolism (membrane potential, oxygen consumption and intracellular ATP levels). Abcam offers a variety of assays to generate mechanism of action data at the individual protein level to further assess the safety of development candidate compounds. These assays measure protein-specific nitration and carbonylation to elucidate effects from oxidative stress, or changes to expression and translocation within the cell of key pro- and anti-apoptopic proteins, or oxidative phosphorylation complexes and ATP synthesis activity (Figure 11).

Stem cells are considered the most relevant cell type for phenotypic assays. Axiogenesis (www.axiogenesis.com) is a leading provider of pure human cell types derived from induced pluripotent stem cells (iPSC) along with assays validated on all relevant commercial platforms. A strong IP position and key licenses enable Axiogenesis to offer FTO in the use of iPSCderived cells including disease models such as cardiac hypertrophy (also hypertrophic cardiomyopathy, HCM). HCM is an abnormal enlargement of the heart muscle ultimately leading to chronic heart failure. A major constraint for the development of adequate therapies has been the lack of suitable cell-based assays with physiological relevance. Axiogenesis, in collaboration with Perkin Elmer, has developed a phenotypic screening assay based on iPSC-derived cardiomyocytes (Cor.4U®) and high content imaging in 96- or 384-well format (Perkin Elmer Opera Phenix™ High Content Screening System). Cor.4U® recapitulates the electrophysiological, mechanical, biochemical and pathophysiological properties of native human cardiomyocytes. They are easy to handle and available in unlimited quantities of reproducible quality. Cor.4U® can be readily induced, through stimulation with the agonist endothelin-1, to reveal a pathological hypertrophic phenotype. This involves an increase in B-type natriuretic peptide (BNP) expression, as well as reorganisation of the contractile apparatus. These effects are dosedependent and quantifiable through high content analysis, combining fluorescent microscopy with automated digital imaging (Figure 12). This assay represents a valuable platform for phenotypic compound screening, both for efficacy and early detection of structural cardiotoxicants. Compared to similar test systems using rat neonatal cardiomyocytes, using Cor.4U® for target identification, primary screening and candidate selection is costeffective and obviates problems of sourcing, handling and cross-species data translation.

While liquid handlers and analysers support highthroughput applications in cell-based phenotypic assays, it is the ability to automate the entire process from start to finish that provides the greatest contribution. The Biomek® family of liquid handlers from Beckman Coulter (www.beckmancoulter.com) enables high-throughput compound, plasmid and microRNA screens by creating an all-encompassing solution through the integration of devices such as plate readers, flow cytometers and high-content imagers. Its SAMI EX software helps manage plate movement in high-throughput applications, while SAMI Process Management efficiently co-ordinates multi-day processes commonly found in phenotypic screens, and DART software ensures data integrity through all steps of the screen. This creates a fully automated system that eliminates user-to-user variability and allows lab technicians to produce and manipulate plates without human hands ever touching a sample (Figure 13). Cytation Cell Imaging MultiMode Reader is BioTek’s (www.biotek.com) solution for both target- based and phenotypic assays. While PMTbased optical detection enables common assay platforms such as ELISA, AlphaScreen, TR-FRET and glow luminescence; CCD-based microscopy enables a wide range of phenotypic assays using both fluorescent probes and/or label-free detection. An example of a tumour invasion assay is provided in Figure 14. In this assay, a co-culture of RFPexpressing human fibroblasts and a GFP-expressing cancer cell line were aggregated into a spheroid in Corning Spheroid microplates, followed by immobilisation in Matrigel containing the chemoattractant CXCL12. After several days of incubation, the extent of cancer cell line invasion into the Matrigel can be quantified by the Cytation’s image analysis software, as evidenced by the gold highlighted perimeter around invadopodia seen in Figure 14. Potent inhibitors of CXCR4 receptor were able to inhibit invasion into the Matrigel in a dose-dependent manner. Many other examples of phenotypic assays conducted with Cytation can be found on BioTek’s microsite: www.cellimager.com/phenotypic.

Over recent years, Cisbio Bioassays has greatly expanded its panel of assays for the monitoring of cellular biological events such as cellular signalling pathways regulation through detection of protein phosphorylation level; quantification of various cytokines or biomarker secretion and activation of cell surface G protein coupled receptors3. Under a lot of circumstances, these HTRF-based assays are used as phenotypic readouts. Taking advantage of a technology approach that was shaped for compound screening, and particularly adapted for cellbased detection, a number of Cisbio’s new assays are now being used by researchers to assess phenotypic modulation of the cells upon compound treatment. For example, Apolipoprotein A1 expression modulation has been proven to be an indicator of various targets upon cell treatment and can be HTS-quantified with HTRF. The upregulation of this protein has been implemented as a readout for two phenotypic screening at GSK and Resverlogix, leading to identification of drug candidates for the treatment of artherosclerosis. The mechanism of action of these compounds was then deciphered and proven to be mediated by epigenetic BET bromodomain/histone interaction inhibition. In a different programme, Phospho STAT3 (Tyr705), one of the phospho-protein assays part of Cisbio’s panel, was a marker used in a phenotypic discovery approach to identify OPB-31121. This compound, currently in Phase II trials as antitumour drug, decreases STAT3 through an unknown JAK-independent mechanism.

More relevant and reliable preclinical cardiotoxicity tests are required to improve drug safety and reduce the cost of drug development. Current in vitro testing strategies predominantly take the form of functional assays to predict the potential for drug-induced electrocardiogram (ECG) abnormalities in vivo. Understandably, multi-electrode array (MEA) assays utilising human stem cellderived cardiomyocytes (hSC-CMs) are becoming increasingly employed as a tool to study functional toxicity, since the data output is analogous to the ECG. Multiple features of this MEA waveform can be analysed to provide a phenotypic profile of a drug’s effects enabling its ion channel blocking activity to be predicted. However, cardiotoxicity can also be structural in nature, so a full and efficient assessment of cardiac liabilities for new chemical entities should account for both structural and functional aspects of cardiac cell biology. GE Healthcare (www.gelifesciences.com) has more than a decade of experience developing high content analysis (HCA) assays for drug discovery with the IN Cell Analyzer system. High-throughput fluorescence microscopy and automated image analysis, in combination with specific probes to key indicators of cardiomyocyte structure such as mitochondrial integrity and calcium homeostasis, provides a multi-parameter approach to the study of structural cardiotoxic liabilities of compounds in hSC-CM models, such as GE Healthcare’s Cytiva Plus. Measuring numerous structural parameters simultaneously provides a phenotypic signature of a drug’s effects which can be analysed using hierarchical clustering techniques to rank compounds by mechanism of action and potential clinical risk. The advent of transparent multi-well MEA plate technology suitable for screening applications has opened up the possibility of multiplexing MEA assays with GE Healthcare’s HCA technology. In collaboration with Axion BioSystems, a leading MEA platform provider, GE Healthcare has recently demonstrated the power of this approach. This multiplexed assay directly links changes in morphology with recorded electrophysiology signatures, offering even greater insight into the wide range of potential drug impacts on cardiac physiology, with a throughput that is amenable to early drug discovery4 (Figure 15).

IntelliCyt’s (www.intellicyt.com) iQue® Screener is the first multiparameter analysis system for screening suspension cells and beads in microplates. By integrating patented instrumentation, software and reagent kits that are optimised for phenotypic screening, antibody discovery and immunology, IntelliCyt has created an automated, benchtop workhorse that can process and analyse microplates in minutes while delivering rich, high content data from a powerful flow cytometry engine. IntelliCyt’s innovations in probe design enable microvolume sampling (1μl) from miniaturised assay volumes as low as 6μl in microplates creating attractive cost savings for reagents and sparing of scarce biological samples. The recent launch of iQue® Screener PLUS measures up to 15 simultaneous parameters to provide superior performance with high throughput. Two exciting examples illustrate the novel synergy of iQue capabilities: i) Phenotypic Drug Discovery leveraging microvolume, high content screening of cells and secreted protein measurements simultaneously; and ii) immunotherapy, with a mixed cell and bead-based assay allowing simultaneous cellular and molecular measurements to be profiled for optimal response, as might be needed for checkpoint inhibition assays. ForeCyt® data analysis and visualisation software rapidly transforms the large, information-rich data sets acquired into actionable results. A screening centric, interactive, assay development, data analysis and result visualisation environment that makes creating highly multiplexed assays, including measuring cells and beads together, intuitive to learn and a pleasure to use. Users can easily assess the data on a per-cell, per-well, or per-plate level, shortening the time to discovery (Figure 16).

Fuelled by cancer research, phenotypic drug discovery experiments have become increasingly more complicated, time-consuming and expensive. With 2D and 3D assays, the management of pick lists, transfer maps, and plate handling protocols to achieve the level of multiplexing desired in highthroughput combinatorial screens is complex and challenging. One step towards simplification is through the elimination of fixed transfer patterns using the any-well-to-any-well capability of acoustic liquid handling. Labcyte Echo® liquid handlers (www.labcyte.com) use focused sound energy to dispense precisely-sized droplets from a source microplate well to assay formats of increasing complexity. The speed at which transfers to complex layouts occurs has made Echo liquid handlers preferred tools in combination screening programmes worldwide. The elimination of plastic tips, reduced sample and reagent use and general ease of use translate to increased productivity and significant cost savings for users. To further improve the ease of adopting Echo systems for combination screening, Labcyte recently released a new software application, Echo® Combination Screen (ECS). The software provides a graphical and user-friendly approach to mapping out the complex transfer patterns required for combinatorial assays or screens. ECS includes a built-in tool for designing dose-response curves that automatically converts starting concentrations and dilution factors into transfer and normalisation volumes for any curve, allowing users to take full advantage of a direct dilution approach. The software supports multi-concentration curves, single concentration transfers, single agent transfers, pick lists and control wells. Together, Labcyte Echo liquid handlers and ECS software enable highly efficient, low cost, targeted identification of biological drug treatment rationale in a fast and robust manner (Figure 17).

The need for biological relevance has driven a shift towards phenotypic screening. The additional complexity involved has generated an increased demand for high-content, high-throughput screening. The ImageXpress Micro Confocal system from Molecular Devices (www.moldev.com) provides higher sensitivity, with advanced illumination techniques for high resolution screening at the speed of widefield5. The complexities of 2D and 3D models can be explored faster and better results gained even in samples grown in a thick extracellular matrices. More physiologically relevant, complex threedimensional models can be screened including spheroids, tissues and whole organisms at high throughput for samples in slides or one to 1536-well microplates. This system provides improved quantification for fixed or live cell assays and can capture high-quality, publication-grade images without sacrificing throughput, reliability or flexibility. AgileOptix technology enables the ImageXpress Micro Confocal system to deliver the sensitivity and throughput needed for demanding applications by combining a powerful solid-state light engine, proprietary spinning disc technology, advanced optics and high-quantum efficiency scientific CMOS sensors. This newest system continues Molecular Devices’ tradition of building capability and throughput into its products, in the last 15+ years, it has increased the throughput of its high-content offering almost 50 fold, enabling researchers to go from 4,000 wells/day to >200,000 wells/day. MetaXpress® Software powers its ImageXpress Micro Confocal system, giving precise control over image acquisition and analysis, all within a unified interface (Figure 18).

iPSCs are essential tools for generating physiologically relevant disease models. Automated systems for iPSC derivation, expansion and differentiation provide cell lines with consistency required for large-scale phenotypical screening assays. Celigo S from Nexcelom Bioscience (www.nexcelom.com) is a plate-based cytometer which can be integrated into an automated iPSC generation process to image and quantify confluence readout for fibroblast growth tracking, calculate doubling time and identify ideal confluence for freezing down in a 6- well plate. It is also used to monitor the growth of fibroblast cell lines post-thaw and before reprogramming. By utilising the F theta lens and galvanometric mirror technology, uniform images are captured rapidly from the entire well, making Celigo suitable for counting TRA-1-60+ iPSC colonies in 96- and 24-well plates. Embryoid bodies (EB) are used for quality control of iPSC lines. Celigo can rapidly image and confirm EB formation in V-bottom 96-well plates. During cell expansion, Celigo is used to obtain live/total cell count from a small aliquot of sample. The data is fed into the liquid handling system for cell passage. Immunohistochemistry staining of differentiated cells in 24-well plate can be imaged and quantified using three fluorescence channels (Ex/Em: 377nm/470nm, 488nm/536nm, 531nm/629nm) (Figure 19).

High content screening (HCS) is one of the core technologies for phenotypic screening as it enables scientists to work even with complex cellular systems including primary cells or 3D microtissues. Depending on the assay, 3D microtissues can be grown in either PerkinElmer’s CellCarrier™ Spheroid ULA plate or in the InSphero Gravity PLUS™ Hanging Drop system. At SLAS 2016, PerkinElmer (www.perkinelmer.com) will introduce the Operetta® CLS High Content Analysis system which, for the first time, combines automated water immersion objectives with stable LED illumination, confocal optics and a sCMOS camera to provide scientists with high sensitivity and flexibility for deep biological insights from cellular samples. The user-friendly Harmony® software controls all aspects of image acquisition and analysis for robust phenotypic fingerprinting with advanced parameters such as texture and STARmorphology. This makes the Operetta CLS system an ideal choice as an assay development tool and for smaller screens. The Opera® Phenix™ High Content Screening system provides unrivalled performance and throughput for large phenotypic screening campaigns with laser excitation, up to four large format sCMOS cameras and its unique confocal Synchrony™ optics. Both systems work seamlessly with Columbus™ software which provides the same phenotypic fingerprinting tools as Harmony software yet in a web-enabled, platform independent, high volume image storage and analysis framework. Results can then be transferred to PerkinElmer’s High Content Profiler™ software for true multi-parametric hit selection through unbiased machine-learning to complete the phenotypic discovery workflow (Figure 20).

Promega (www.promega.com) offers a wide range of assays for phenotypic drug discovery, including a broad portfolio of cell viability and cytotoxicity assays. It recently expanded its focus on real-time monitoring of cells with the RealTime-Glo MT Cell Viability Assay, which allows researchers to monitor cells out to 72 hours. Toxicity is both dose- and time-dependent; therefore, the ability to monitor cell viability changes in real time is a needed improvement over traditional endpoint assays. RealTime-Glo uses the bright and versatile NanoLuc luciferase enzyme along with a cell-permeable pro-substrate. Once the components have been added to cell cultures, only viable cells reduce the pro-substrate to an active form for use by the luciferase present in the medium. The reduced probe is rapidly used up by luciferase and does not accumulate. Changes in cell viability can be detected immediately (Figure 21). This time-dependent measurement allows analysis of drug potency versus efficacy and cytostatic versus toxic drug effects. It also informs decisions about the timing of treatments and the use of other functional endpoint assays. RealTime-Glo is non-lytic, homogeneous and well-tolerated by cells making it ideal for drug screening applications such as phenotypic screening. Cells are available for further analysis, after being treated with the RealTime-Glo reagents, such as with multiplexed assays or other downstream applications (eg qRT-PCR). Because the RealTime- Glo signal decreases rapidly upon cell death, one can multiplex many assays with lytic components on the same test samples, including other luminescent assays, without the need for spectral filters. With the ever-escalating costs of bringing new drugs to the market, it is vital to minimise the risk of late stage failures due to poor efficacy or off-target activities. Phenotypic screening has a central role to play in this, and Tecan (www.tecan.com) is a market leader in laboratory automation for cell biology applications. The company’s recently launched Fluent™ laboratory automation solution for cell-based assays has a broad range of features specifically designed to enhance throughput, streamline workflows and deliver more precise and reliable results. It helps to optimise the performance of cell-based assays by simplifying the handling of precious samples and seamlessly integrating with cell biology devices, such as HEPA hoods, wash stations and readers. Tecan’s Spark® 10M multimode plate reader has also been developed with cell-based assays in mind, with an integrated Gas Control Module (GCM™) offering automated regulation of CO2 and O2 concentrations within the measurement chamber to ensure a stable, longterm cell culture environment. It also features a built-in cell counter which can analyse a broad range of cell sizes and types, providing fully automated, label-free cell counting and analysis. Together with evaporation protection and automated microplate lid handling, this instrument ensures ideal conditions for live cell assays. Tecan is also working closely with suppliers of three dimensional cell culture technologies – such as Lonza (RAFT™ matrix), Reinnervate (Alvetex®) and InSphero (3D InSight™) – to offer complete automation of 3D cultures, allowing primary and stem cells to be grown in an in vivo-like environment for greater biological relevance (Figure 22).

When you embark on phenotypic drug discovery with Thermo Fisher Scientific™ (www.thermo. com) you are tapping into a legacy of innovation going back to the origins of high content analysis. That legacy has helped to generate more than 1,000 publications in the field of ‘cellomics’ since the introduction of ArrayScan™ HCA readers in 1999 and more than 50,000 publications using Invitrogen™ Molecular Probes fluorescent reagents. Maintaining its leadership role, Thermo Scientific™ recently introduced the CellInsight™ CX7 high content screening platform as an integrated system for all-round performance in phenotypic drug discovery. The system offers a choice of imaging modes with seven spectral channels to extract the information you need from your samples. Use the entire fluorescence spectrum to multiplex your assay, and select either widefield or confocal optics for any channel. You also have fourcolour brightfield options for colorimetric analysis of tissue sections. Invitrogen fluorescent reagents are the preferred tools for the labelling and detection of cellular targets while Thermo Scientific HCS Studio™ Cell Analysis Software helps you combine those components and build applications to answer your phenotyping questions. Invitrogen offers the most complete suite of complementary reagents to interrogate cell health and measure parameters such as viability, proliferation, apoptosis, hypoxia, autophagy and reactive oxygen species. Additionally, Alexa Fluor™ dyes provide the brightest and most photostable labels for specific protein, organelle, or cell detection. In the Thermo Fisher Scientific portfolio you have the multiplexing tools to see cellular phenotyping in its most complete biological context (Figure 23).

In order to maximise the benefits of phenotypic screening and minimise the chance of missing a hit, it is beneficial to screen against a full compound library rather than library subsets, using high-content assays. Despite the benefits, such an approach is challenging using an automated high-content microscope as HTS requires: very rapid throughputs, miniaturisation to minimise cell and reagent costs, a simple approach to hit identification and manageable data output. TTP Labtech’s (www.ttplabtech.com) acumen Cellista reduces these challenges by providing the value of a highcontent approach for hit identification in a format that is approachable to HTS. With throughputs of more than two million data points a week, cellular imaging at high-throughput is achieved by laser scanning excitation through a specialised F theta lens and photomultiplier tube (PMT) signal detection. This design enables rapid whole-well imaging for multiplexed high-content assays, including 3D spheroid models due to its large depth of field. Recent improvements to Cellista software has been developed in response to the needs of an efficient screening laboratory. Workflow simplification improves productivity via a rapid and straight forward approach to data acquisition, analysis and validation. Cellista software removes the adoption barriers associated with image analysis through a eliminating the requirement for specialised operators. Data output files are small, enabling acumen Cellista to be integrated into a screening workflow with little change to existing infrastructure. Full library phenotypic screening can now easily be achieved in high-throughput using a high-content approach with acumen Cellista (Figure 24).

Yokogawa E lectric Corporation (www.yokogawa. com/scaner) offers two types of phenotypic screening imagers with its microlens-enhanced spinning Nipkow disk confocal technology (Yokogawa CSU), by which photo-toxicity and photo-bleaching are drastically reduced, making the systems ideal for use in observing live cell, but also good at capturing clear-cut Z-stacks with high speed. The CQ1 has a true confocal optics and environment control module in spite of desk-top compact size. The performance of CQ1 is comparable to the high end high-content imagers. It brings 3D image acquisition and 3D on-the-fly analysis for 3D samples such as spheroids, and draws the charts of the results. CQ1 addresses a wide range of imaging samples from live cells to tissue sections with microplate up to 1536-well format, 35mm dish, 60mm dish, slide glass and coverglass chamber. CV7000S is a high-end HCA system with multiple cameras which can be used for highthroughput HCA screening. Both the CQ1 and CV7000S have stage incubators, which makes it possible to acquire long-term time lapse imaging. As its maximum image acquisition rate is 38 fps (at 3X3 binning of camera pixels), fast phenomenon in live cell is observable. In addition, its unique optional build-in dispenser enables kinetics imaging before and after cell treatment with reagents. For example, beats of iPS cell-derived cardiomyocytes can be clearly observed with a Ca2+ probe. The experiment is applicable for testing irregular heartbeats and toxicity for drug discovery. CV7000S can be installed in up to a total of six objective lenses, including a 60x water immersion lens with automated water supply, and is capable of high-resolution imaging available with its Yokogawa CSU (Figure 25).

Discussion
Table 1 summarises the technologies facilitating phenotypic screening assays reported in the vendor updates above. The majority of these updates relate to the use of high content analysis systems enabling multi-parametric analysis of cellular phenotypes with quantitation obtained through automated image processing. Of note here is the emphasis on increased sensitivity CMOS cameras, high resolution confocal optics and higher throughput capability of the imaging systems to derive greater information content from larger phenotypic screening campaigns (GE Healthcare, Molecular Devices, PerkinElmer, Thermo Fisher Scientific, Yokogawa Electric). These imaging systems are not only suited to adherent cultures, but are also increasingly optimised to explore the complexities of 3D culture models, including spheroids and microtissues. Other systems based on automated flow cytometry (eg Intellicyt) derive similar information-rich multiparametric data from screening suspension cells. Several imaging cytometers enable rapid whole-well imaging for multiplexed high content screening, including 3D spheroid models due to their large depth of field (Nexcelom, TTP Labtech). CCD-based microscopy enabled on a multi-mode plate reader can also support a wide range of phenotypic assays using both fluorescent probes and/or label-free detection (BioTek). Several vendors of automated liquid handling and robotic workstations (Beckman Coulter and Tecan) now offer systems fine-tuned to the complexities of the more relevant cellular models required in phenotypic screening. Other liquid handlers (Labcyte) uniquely support the setup of drug combinations of particular relevance to phenotypic cancer screening. Human stem cells are considered the most relevant cell type for phenotypic assays, and the commercial availability of an ever-increasing list of cell types, together with advances in detection technologies, is fuelling the development of many novel highly pertinent disease models (Axiogenesis, GE Healthcare). Finally, we should not ignore the many reagents and assay kits, previously used extensively to support real time monitoring of cellular biological events in target- based drug discovery, have a role to play as phenotypic readouts and in some cases facilitate multiplexing of cellular phenotypes or the development of counter screens such as cytotoxicity and pathway selectivity (Cisbio, Promega, Thermo Fisher Scientific).

Table 2 lists some of the feedback we obtained from survey respondents on the biggest unmet needs in PDD and the new tools that are required to drive the investigation of phenotypic screening assays. One aspect that stands out as an ongoing challenge in PDD is identifying the molecular targets of active hits from phenotypic screens. This target deconvolution is a crucial process that is required to understand underlying mechanisms and to further optimise active compounds. Of the many approaches investigated, the use of annotated compound sets, prepared specifically for phenotypic screens, plus target enrichment analysis has aided the delivery of the target hypothesis rapidly in some Pharma, although it is widely claimed that new gene editing techniques such as CRISPR/Cas9 knockdowns are set to revolutionise target deconvolution. The reality is that multiple technologies have a role to play in target deconvolution, just as multiple approaches, of which PDD is just one, all have a role in successful drug discovery.


Dr John Comley is Managing Director of HTStec Limited, an independent market research consultancy whose focus is on assisting clients delivering novel enabling platform technologies (liquid handling, laboratory automation, detection instrumentation; assay methodologies and reagent offerings) to drug discovery and the life sciences. Since its formation 14 years ago, HTStec has published 121 market reports on enabling technologies and Dr Comley has authored 56 review articles in Drug Discovery World. Please contact info@htstec.com for more information about HTStec reports.

References
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2 Phenotypic Drug Discovery Trends 2015. Published by HTStec Limited, Godalming, UK, September 2015.

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4 Clements, M et al (2015). Bridging Functional and Structural Cardiotoxicity Assays Using Human Embryonic Stem Cell-Derived Cardiomyocytes for a More Comprehensive Risk Assessment. Toxicol. Sci. 148(1), 241-260.

5 Sirenko, O et al (2015). High-Content Assays for Characterizing the Viability and Morphology of 3D Cancer Spheroid Cultures. Assay Drug Dev Technol. 13(7): 402-414.