• In the previous article of this two-article set, we presented the current status of the high-throughput screening (HTS) industry in terms of the qualitative trends and drivers and the quantitative metrics that characterize current screening operations. We focused in that article primarily on the size of screens, the numbers of data points on average that are generated, as well as the library sizes that are screened. Much of the information was derived from the ScreenTech World Summit 2003 held in March 2003 in San Diego, CA.
• In this, the second article from the ScreenTech conference, we focus upon the technology trends in this space as they relate to the different drug target classes of relevance in drug discovery, and how these emerging technologies are expected to impact HTS going forward.

Introduction and Overview of Emerging Technologies in HTS from the ScreenTech Conference
The 2003 ScreenTech World Summit was a multitrack conference, whereby different topics and drug target classes of value in HTS were presented in parallel. It is becoming clear that the hitherto amorphous HTS industry is now becoming gelled, in that certain technologies and approaches have established themselves as the workhorse of the industry, and there are emerging technologies and trends on the horizon. In this section of the article, we address some of the preeminent emerging technologies in the space and discuss each in terms of its value in the HTS arena.

Emerging Technology in Multiplexing of Cell-Based Assays for HTS: Using Nonpositional Arrays
A number of companies presented technologies in this area. Virtual Arrays (Sunnyvale, CA, USA) presented their digital bar code-based technology for addressing targets in cells In Vivo, in multiple cellular populations simultaneously. This multi-parametric, multi-cell analysis is a powerful enabling tool for the analysis of drug targets as well as toxicity markers in single wells. The presentation by Virtual Arrays at the conference demonstrated how G protein-coupled receptors as well as absorption, distribution, metabolism, excretion (ADME)/toxicity markers could be simultaneously analyzed in a cell-based format. A schematic of the digital bar codes from Virtual Arrays is presented in Figure 3 (note that each of these bar codes--nanocarriers can accommodate one cell type, and the technology currently enables 10-50 different cell types to be interrogated simultaneously).

Detection System for HTS that Involves Enzymatic Amplification of the Readout Signal
DiscoveRx Corporation (Fremont, CA, USA) presented its technology platform for cell-based assays utilizing the complementation of enzyme fragments from ß-galactosidase as a readout system. This technology is in line with the current theme in cell-based assays whereby key receptors (on the cell surface) as well as intracellular molecules of value in signal transduction are labeled using a biological tag. In the case of the DiscoveRx technology, the tag is a fragment of the ß-galactosidase enzyme that "tracks" the biological pathway under interrogation. The use of an enzymatic tag enables a very sensitive readout of the biology in question (such as G protein-coupled receptor [GPCR] signaling, for instance). This is because very small signals (emanating from the biological event in question) are amplified as the reconstituted enzyme, ß-galactosidase, and turn over substrate into product--hence, this technology is a biological signal amplification system for intracellular events. Figure 4 schematically presents the DiscoveRx technology platform and how the ß-galactosidase system is an amplifiable marker for cell signaling In Vivo.

Labeling Signaling Pathways with Reporters In Vivo for Screening
An interesting technology for cell-based assays of important pharmaceutical targets was presented at the conference by Bionaut Pharmaceuticals (Cambridge, MA, USA). Bionaut is championing a technology whereby reporter genes are introduced into targeted genetic sites (within key genes in cells In Vivo). In this manner, the company creates what they term as Sentinel Lines(TM), which enable dissection of signaling pathways In Vivo. In other words, a reporter gene can be delivered into every genetic site regulated by insulin. Each cell clone isolated using this approach can then be used to monitor activity of insulin or screen for new drug candidates--whether they stimulate or inhibit targeted insulin pathway(s). In a sense, this is a molecular version of the traditional pharmaceutical approach--create a disease model and then screen drugs against it to see what works. The isolation and use of multiple cell lines, each embodying a unique reporter gene, allows early and rapid screening for pathway redundancy, specificity, and adverse side effects--issues responsible for the substantial portion of drug failures during downstream clinical trials. It will be interesting to note how well the pharmaceutical industry embraces the Bionaut approach to cellular pathway interrogation.

Multiplexed Assays using Positional Arrays in Microtiter Plate Format
A very interesting technology for multiplexing was presented by Meso Scale Discovery (MSD; Gaithersburg, MD, USA). This company is focused on the use of electrochemiluminescence detection readout coupled with the use of arrays in the bottom of traditional SBS-footprint microtiter plates to enable HTS across a number of assay methodologies (in either biochemical or cell-based format), such as:

• Immunoassays.
• Receptor ligand interactions.
• Transcriptional assays.
• Protein translation assays.
• Protein nucleic acid analysis.

The technology from MSD utilizes traditional microtiter plates (96-, 384-, or 1536-well footprint) that work in combination with custom plate bottoms, which essentially is a sheet with integrated electrodes. In this manner, each well in a plate has multiple "addressable" sites on its bottom. The detection system is an imaging-based system that reads chemiluminescence emitted by the plate upon stimulation by the electrodes (below the plate). MSD has built a high-throughput instrument that accommodates the plate(s) and the electrode plate beneath it. In addition, it has a charge-coupled device (CCD)-based imager with a telecentric lens that enables the plate to be imaged en bloc. The MSD system for screening enables a wide range of applications (i.e., complex assays for HTS) to be run on it, such as:

• Kinase assays.
• Ubiquitination assays.
• Receptor ligand-binding assays.
• GPCR assays (binding-based and functional).
• Multiplexed assays (for cytokines and for autophosphorylation).
• Immunoassays in cell lysates.
• Enzyme-linked immunosorbent assay (ELISA) replacements (antibody selection).
• Integrins.
• DNA-protein-binding assays.

Similar to the Virtual Arrays technology platform, the MSD technology enables, e.g., multiplexed analysis of GPCR drug targets. In summary, MSD made a very strong showing at the ScreenTech conference where they presented their technology platform, which included the following:

• Instrumentation that is screening-ready, is in nonwashing format, delivers sensitivity, and dynamic range.
• Plate-based assays.
  • Multispot microplates (for multiplexing).
  • Compatible with existing automation for screening.
  • Reagents allow rapid assay development.
• Complex biological assays can be run deploying this technology.
• Membrane-based assays for GPCRs, epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and whole cell assays.
  • Kinases.
  • Ubiquitinylation.
  • Multiplexed assays.
  • Integrin assays.
  • DNA-protein-binding assays.

Amgen (Thousand Oaks, CA, USA) at the ScreenTech conference presented a technology assessment of the MSD technology platform. The conclusions from the study were that the MSD technology platform enables the building of assays quickly, the identification of hits, potency and selectivity assignment to these hits, and determination of cellular efficacy. In brief, the ability of the MSD technology to perform the widely accepted biologies in the HTS space coupled with the ability to multiplex assays using conventional instrumentation makes MSD a powerful entrant in the HTS space.

Addressing Protein Kinase Targets in HTS
The emerging technologies presented earlier in this article are applicable to a number of different target classes in drug discovery. For instance, the Virtual Arrays technology can be applied to a number of different target classes, although it appears to work well with cell-associated targets, such as GPCRs. Some target classes, such as the protein kinases, can be screened effectively in both biochemical and cell-based systems, and the need in such a case is to be able to develop multikinase assays that can address a large number of kinase targets In Vivo.

Protein kinases are an important target class for drug discovery, since protein phosphorylation is a central element in cell signaling. Thirty percent of all proteins contain covalently bound phosphate, 518 protein kinases and about 100 protein phosphatases are encoded in the human genome (assuming a relatively conservative number of 100,000 proteins, each kinase phosphorylates more than 200 proteins In Vivo). Also, it is known that mutation or misregulation of protein kinases is the consequence of many disease states, hence it is of value for the pharmaceutical industry to be able to screen protein kinases efficiently in a high-throughput manner.

Many cancers demonstrate up-regulated protein tyrosine kinase activity, as presented in the Table 2. The purpose of Table 2 is to demonstrate how key kinases are in human cancer and thereby offer an economic opportunity to the pharmaceutical industry for drug discovery and development, against this target class.

Upstate (Charlottesville, VA, USA) presented at the conference their technology and scope of assays that enable various human kinases to be interrogated. Upstate presented its lead profiling business, KinaseProfiler(TM), which is composed of enzymes that have therapeutic relevance, address the diversity of kinases, and cover subfamilies of kinases. The panel of kinase assays comprises about 70 different assays covering a large amount of the known kinase real estate In Vivo. Using this approach, general kinase inhibitors can be quickly identified and further characterized. Subsequent to this initial identification, a selectivity profile can be performed to identify selective kinase inhibitors (e.g., those of the Src family of nonreceptor tyrosine kinases). Selectivity profiling enhances kinase drug development as it quickly defines potential toxicology issues, which can have a triage role in the drug discovery process.

Survey of the Landscape of Emerging Fluorescence-Based Assay Technologies in HTS
A number of vendors at the ScreenTech conference presented new fluorescence-based technologies and gave presentations together with the pharmaceutical and biotech end-users. Jeff Stack of Vertex Pharmaceuticals (Cambridge, MA, USA) delivered an excellent presentation that focused on some of the key state-of-the-art technologies in HTS that Vertex is deploying in its drug discovery efforts.

The Vertex San Diego screening facility is based upon the Aurora Biosciences acquisition, and this facility has miniaturized screening to the 3456-well format (the well-known Aurora Nanoplate format). In addition, the Aurora facility has a dedicated HTS line for ion channel targets and a fully automated compound handling system for rapid turnaround of hit picks and dose-response titrations.

Vertex possesses some of the most current fluorescence-based assay technology portfolio for screening, such as:

• GPCR screening using ß-lactamase reporter gene system.
• Cytokine receptor screening using ß-lactamase reporter gene system.
• Nuclear receptor screening using ß-lactamase reporter gene system.
• Ion channel screening using voltage-sensitive fluorescent probes.
• Cytochrome P450 assays.
• Protease target assays using green fluorescent protein (GFP), fluorescence-resonance energy transfer (FRET) peptides, and ß-lactamase reporter gene system.
• Kinase and phosphatases target screening using FRET peptides and cell-based assays.
• Protein translocation (redistribution) assays In Vivo, using GFP.

The above technology portfolio covers the majority of the emerging assay biologies in widespread use across the pharmaceutical screening landscape. Indeed, the use of ß-lactamase reporter gene systems is widespread as a fluorescence-based system for functional assays of target classes, such as GPCRs and nuclear hormone receptors (NHRs). GFP is a very well-established detection system for the readout of biological translocation events In Vivo and more recently has begun to be utilized as a protein sensor In Vivo. Spectrally distinct GFPs are available from a number of vendors that can be used to put together FRET assays for biologically relevant events In Vivo. In addition, this portfolio of technologies includes fluorescent probes that are FRET-based with voltage-sensing properties, and therefore are of value in ion channel high-throughput screening.

The ß-Lactamase Reporter Gene System for Functional Screening: A Technology Platform for Addressing the Key Pharmaceutical Targets in a Physiologically Relevant Context
Much of the technology presentations at the ScreenTech conference were focused upon functional assays for HTS, given that receptor-ligand interactions and their perturbation per se by small molecules do not convey enough biological information for effective screening. The emerging technologies for screening all seek to functionally interrogate (drug) targets using intracellular readouts. These technologies all necessitate the use of intracellular sensors of biological functionality, such as GFP and ß-lactamase, among others.

The ß-lactamase enzyme has a specific activity In Vivo. It converts a substrate that is fluorescent to a different colored product that can be measured using fluorescent microscopes, flow cytometric cell sorters, or simply fluorescence-reading plate readers.

The value of the ß-lactamase system is that it can be used as a biological readout for the activation of a signal transduction pathway In Vivo, which is stimulated upon activation of cell surface receptors, such as GPCRs. The signal transduction pathway activated by the receptor (interacting functionally with its ligand), triggers the production of ß-lactamase enzyme In Vivo that then cleaves the substrate, which is a cell-permeable fluorescent molecule, into a product that can be spectrally distinguished from the substrate.

The power of this technology stems from the fact that cell lines can be developed containing the reporter system (the ß-lactamase gene under the control of the appropriate regulatory elements) and these can be selected using flow cytometry. These "hard-wired" cell lines can then be used for a variety of assays addressing target classes such as GPCRs, proteases, kinases, and nuclear receptors. Note that since the fluorogenic substrates are cell-permeable, there is no need for cell lysis, and intact cells can be analyzed in situ. In addition, the homogeneous nature of this assay lends itself well to automation.

Another powerful feature of this reporter system is that since the fluorescent measurements are ratiometric, this internally controls for experiment-to-experiment variations in reagents, cell numbers, and other environmental conditions.

In summary, the reporter gene-based systems have value as a screening technology platform since they provide a functional readout for the target class that they interrogate. Perhaps, a concern in utilizing the technology is how tightly the activation of the target (under interrogation in the screen) is with the triggering of the reporter gene function.

Imaging in HTS
A technological move in the HTS landscape is the shift towards imaging and live-cell (intact) assays in screening. This is, in part, driven by the need to develop and deploy screening technologies that can quickly triage the nondruggable from the druggable targets. The technologies and instruments presented at ScreenTech were focused on live cell assays and approaches for high-content screening.

In this vein, companies such as Atto Bioscience (Rockville, MD, USA) presented their live cell-imaging platform that enables intact cell-based assays to be performed. Also, Amersham Biosciences (Piscataway, NJ, USA) presented their IN Cell Analyzer instrument platform, which enable high-throughput high-content screening in a cell-based format. Therefore, assays for translocation of nuclear factor kappa B (NFkB) from the cytoplasm to the nucleus can be run on such an instrument, and assays for the translocation of moieties from the cytoplasm to the cell membrane (or vice versa) can be performed. The instrumentation in essence is a high-resolution fluorescent microscope that is microplate compatible (96- and 384-well format) that images the wells and uses powerful image analysis to interrogate the data so generated. Using such an instrumentation platform, and the intellectual property rights around GFP, Amersham Biosciences is building a portfolio of assays for some of the key signal transduction molecules In Vivo and following up with commercial products, such as stable cell lines, vectors, as well as validated assays. This menu of products for HTS is the direction in which this marketplace is heading.

Technology Trends in the Screening Space Going Forward
This article together with its companion (the previous article in this two-part set) addresses the technology trends together with the quantitative metrics of the HTS space.

The following list presents our assessment of technology trends in screening going forward:

• Increasing emphasis placed on the assay biologies for screening (both primary and secondary).
• Decreasing emphasis on detection methodologies and readouts--standardization around particular detection platforms as well as instrumentation.
• Increasing utilization of intact cell-based assay technologies across the screening landscape for the analysis of key signaling molecules In Vivo, such as the interrogation of NFkB, inhibitor kappa B (IkB), Janus kinases (JAKs), signal transducers and activators of transcription (Stats), and other signal transduction intermediates.
• Decreasing emphasis on purely receptor-ligand binding assays and an increasing emphasis on functional assays that give a readout based on the triggering of the biological event (such as receptor activation with ligand).
• Increasing emphasis on technologies where the target screening is coupled to a toxicity screen, such that toxic compounds can be quickly triaged out of the screening process.
• Increasing emphasis on multiplexed assays, whereby either multiple different cell lines or different targets are all interrogated in a given well of a microtiter plate, thereby reducing the cost of precious reagents and increasing the speed of the screening ensemble.
• Increasing the emphasis on technologies that can transcend individual target classes, which can be utilized for screening across lots of different target classes.