Keynote Presentation
Recent Advances in Biosensors
Christopher R. Lowe, University of Cambridge, UK

Many extravagant claims have been made in the past two decades about the likely impact of biosensors on the diagnostics industry: In reality, the current market size is still a minute percentage of the global diagnostics market. This presentation looks at the need for, and advantages of biosensors, the technology that has been developed over the past two decades to address these perceived needs and describes newer technologies which may circumvent the problems and suggests how they are likely to have a much more significant impact in the future, particularly in sectors such as diagnostics and drug discovery.

Rapid, Label-Free Screening & Affinity Ranking of Fragment Libraries Binding to Drug Targets
Stefan Löfås, GE Healthcare Biosciences AB, Sweden

Label-free interaction analysis based on surface plasmon resonance (SPR) allows detailed investigations of molecular binding events and has therefore become a standard tool for academic research and drug discovery. Since the introduction of the technology almost two decades ago, BiacoreTM systems have been used to determine real-time kinetic, affinity and specificity data for binding interactions.

There is now an increased focus on finding privileged structures in small molecule drug discovery as new lead-finding screening strategies are introduced. Fragment based lead discovery is becoming widespread to identify suitable scaffolds. Biacore systems have been proven suitable for fragment screening and selection, with a throughput of >1,000 fragments/day using only a few micrograms of target protein. It has been recognized as an important primary screening technique for use as the first step in the prioritization for structural determination. The technology also provides information on the selectivity of binding and target site specificity, which are important criteria in fragment selection, and can also be used for fragment library cleanup. As well as affinities in the mM to pM range, the residence time (off-rate) can also be measured. The presentation will describe optimized workflows and examples of this highly suitable label-free technique for screening and selection of fragments.

Evolution of Label-Free Screening on the Corning® Epic® System
Martin Coldwell, Kathy Dodgson and Phil Rawlins, AstraZeneca R&D Charnwood, UK

Examples of label-free screening at various stages of the HTS cycle – fragment screening against a soluble protein, comparison of a 100K compound HTS in label-free and functional screening will be described. In addition, proof of concept studies for potential future screening modes will be shown – endogenous receptor counter screening in primary cells, solubilized GPCRs, label-free protease assays.

Rapid and Versatile Characterization of Small Molecule Kinetics using Bio-Layer Interferometry
Eric Martin, Novartis, USA

BioLayer Interferometry (BLI) provides label-free, real-time measurements from proteins to small molecules for kinetics characterization and screening. Affinities can be measured ranging from pM to mM with association and dissociation rates for better characterization and selection of drug candidates. Good agreement was found was found with SPR, ITC, NMR, and high concentration biochemical screening methods for small molecules down to MW=150. Applications included fragment-based screening, screening of inactive enzymes, and protein quantitation in cell lysates.

Fragment-Based Screening for Lead Identification & HTS Hit Validation Using Surface Plasmon Resonance Based Biosensor
Tony Giannetti, Roche, USA

Advances in surface plasmon resonance (SPR) biosensor technique have allowed applications of the technology, such as that implemented with Biacore instruments, to extend beyond protein-protein interactions to small molecule-protein interactions in the area of hit finding and hit validation.  For hit finding we have combined the latest methodologies in SPR technique with the techniques of traditional high throughput screening to develop a rapid procedure for hit identification from low molecular weight compound fragment libraries (MW < 300).  Key features of the assay are the ability to screen and verify ~5000 compounds in a few weeks, large dynamic range of the assay (100 pM to 5 mM) and low amounts of protein used (< 0.5 mg from assay development through hit validation).  Case examples of SPR-based hit identification, co-crystallization, and medicinal chemistry development of fragments will be presented.  For hit validation we use the SPR experiment to identify promiscuous inhibitors; compounds that achieve enzyme inhibition through a non-specific aggregation-type binding mechanism.  These compounds are a source of false-positive hits in biochemical inhibition assays, such as those used in HTS, and need to be removed from screening hit lists since they cannot be developed into drug candidates by medicinal chemistry.  The SPR assay can be used to rapidly identify these compounds from large hit lists.  Additionally, the time resolution of the assay has allowed us to identify a variety of mechanisms of action these molecules employ to inhibit protein function.

A Novel Multiplex SPR Array System for Rapid and Efficient Screening of Label Free Binding Assays
Tsafrir Bravman, Bio-Rad Laboratories, USA

Surface Plasma Resonance (SPR) is widely used for kinetic studies of interactions between label-free bio-molecules in real time.  The ProteOn™ XPR36 protein interaction array system (Bio-Rad Laboratories) is a multiplexed SPR system providing detailed kinetic profiles of a 6 by 6 array of molecular interactions in a single injection experiment.  Multiplexing improves and expands the capabilities of traditional SPR technology and workflow by enabling multiple quantitative protein binding experiments in parallel. The interaction of multiple leads with a target protein can be measured simultaneously.  Because multiple conditions can be tested in parallel, robust kinetic analysis of an analyte concentration series can be handled in one experiment without the need for regeneration. In the area of monoclonal antibody (mAb) therapeutics development, significant work involves the production and purification of monoclonal antibodies with very high affinity for the intended ligands, as well as very high specificity and low cross-reactivity.  Current methods for screening potential mAb candidates are slow and expensive, usually requiring purification of the antibody from the hybridoma supernatants before screening can occur.  These methods can also require time-consuming and expensive preparation of labeled ligands, with the added risk that the presence of the label will alter the interaction between the antibody and its ligand.  The ProteOn XPR36 is an ideal tool for reducing the amount of time required for the screening process and offers simple, direct measurements of the protein interactions.

In this presentation, we will discuss the advantages of using the ProteOn system in three application areas to demonstrate robustness of data and significant time-saving afforded by the system: 1) Rapid screening of large hybridoma libraries for the development of diagnostic antibodies; 2) Screening and affinity maturation studies of tumor targeting antibodies; and 3) Small molecule lead identification and optimization.  Our results demonstrate that a multiplex SPR array system such as the ProteOn XPR36 enables label free protein dynamics to be elucidated in hours instead of days.

Finding Needles in Haystacks - in vivo Drug Metabolism
Russell Mortishire-Smith, J&J PRD, Belgium

The study of the metabolism of a compound plays a key role in drug discovery and development, and contributes to the optimization of drug candidates and to the support of toxicity studies. However, the identification of metabolites in complex biological matrices, particularly in vivo, is challenging due to the presence of a large excess of endogenous compounds that interfere with the detection of drug-derived material generally present at relatively low concentrations. Specificity in characterizing drug metabolites can be achieved via radiolabelled drugs, but these are rarely available in the discovery phase of a project. Accurate mass and isotope filtration-based techniques offer valuable alternatives, which are scalable, generic, and relatively easily applied.

SPR Screening of Chemical Microarrays: Advantages and Applications
Renate Sekul, Graffinity Pharmaceuticals GmbH, Germany

Surface plasmon resonance (SPR) has become a well established technique for studying biomolecular interactions in a sensitive and label-free format. Graffinity´s SPR imaging of chemical microarrays can generate affinity data in a high throughput mode Its high detection sensitivity allows the identification of weak interactions and is therefore particularly suited for fragment screening. The talk presents key features and applications of Graffinity´s SPR screening technology.

Bridging the Affinity Gap: Application of Label-Free Methods in Fragment-Based Lead Generation
Stefan Geschwindner, AstraZeneca R & D, Sweden

Even though high-throughput screening (HTS) is seen as the most common approach within the drug discovery process, there remain fundamental issues that limit its scope. Fragment-based lead generation (FBLG) addresses most of those issues and is increasingly being accepted as a valuable complement to HTS.

The FBLG process roughly consists of three phases and starts usually by screening small collections (100s-1000s) of low MWt compounds (150-250 Da) against the target of interest. In the following expansion phase the resulting Hits are analysed and nearest neighbours (analogues) are tested with the aim of increasing affinity and developing preliminary SAR. Finally, synthetic chemistry is applied to further derivatise and optimise the best ligands, usually supported by structure-based drug design. In that context the presentation will focus on the application of label-free methods as a tool to bridge the existing affinity gap between an conventional HTS-like assay and very sensitive methods like e.g. NMR in order to deliver decisive data on affinity and MOA of primary fragment hits and their analogues. The concept will be presented on an active drug discovery project, where FBLG was applied to find novel small-molecule inhibitors of ß-secretase (BACE), a key target for the treatment of Alzheimer's disease.

As the limitations in the affinity range and analyte masses are usually not well-suited for the direct binding analysis of small and weak-binding fragments using SPR technology, an inhibition in solution assay (ISA) was established to study those weak interactions using BIAcore technology. In order to assess the pH-dependent binding of those fragments to BACE, a thermal-shift assay has been applied that helped to identify required changes in the crystallization system and thus increased our ability to obtain structural information with fragments. As a result of that work, several interesting fragment hits coming from a 1D NMR screen could be characterized using the BIAcore ISA and suitable candidates could be soaked into protein crystals that helped the rational design of more potent hit analogues resulting in lead molecules with high affinity and desired biological.

High-Throughput Mass Spectrometry for Label-Free Enzyme & Cell Assays
Philip Tagari, Amgen, USA

Compound profiling for selectivity within a family of related enzymes can present numerous challenges when using traditional fluorescence-based assay approaches. Fluorescence detection reagents typically need to be developed for each enzyme/substrate pair, which can be labor intensive and time consuming. Furthermore, selectivity profiling using the same assay format may not be feasible, or require nonphysiological substrates, if the targets to be profiled act on vastly different molecular classes of substrates. In this study, quantitative high-throughput mass spectrometry using the RapidFireTM platform was developed for compound profiling across related enzymes that utilize a wide range of substrates including, peptides, oligonucleotides and lipophilic small molecule metabolites.

Cell-Free-Electrophysiology – A Label-Free, High-Quality Readout Technology for Transporter Drug Discovery
Henning Vollert, University of Kiel, Germany

Being ubiquitously expressed in all tissues and cell types and being directly involved in manifestation of various diseases, transporters are important targets for pharmaceutical research. Moreover, pharmacokinetic behavior of many drugs is influenced by transport protein activity. Yet, technical limitations of available technologies in the past have lead to an under-representation of transporter targets in pharmaceutical laboratories. Recently, a new technology platform – cell free electrophysiology – became available. We tested this so-called SURFE2R Technology (SURFace Electrogencic Event Reader) and compared the generated data with data produced by conventional technologies. Two transport proteins served as test systems: the sodium / calcium exchanger NCX1, which is assumed to be an important target in cardiomyocytes, and PepT1, which allows b-lactam antibiotics to enter the organism through the intestinal epithelia, thus being of importance for bioavailability issues.

The new technology utilizes a fluidic system to produce substrate concentration jumps above a biosensor surface, which exposes immobilized membrane entities with the protein of interest to the bulk solution. The sudden change in substrate concentration provokes a detectable charging current, which reflects the onset of the transport activity. Direct high-content functional information is thus accessible without compounds other than substrates, superseding the need for labelled substances.

IC50 values of reference compounds obtained with SURFE2R measurements of NCX1 compared well with conventional patch clamp results. They were, however, generated in just a fraction of the time needed for the more conventional patch clamp experiments, and required a much lower level of skills. Data quality of the SURFE²R measurements was far superior in terms of precision and reproducibility. This outcome motivated the decision to use the new technology routinely for NCX1 screening and compound profiling tasks, leading to the successful identification of new inhibitors.

Furthermore, a PepT1 assay has been used to investigate the mode of action of a number of compounds, which has so far only been shown to inhibit substrate activity competitively. Based on the outlined results, we came to the conclusion that cell free electrophysiology in a label-free format is a versatile tool for several applications within the whole drug development process. Secondary screening and compound profiling will benefit from this technology just as much as ADME studies.

Automated Patch Clamp for High-Quality, Versatile Ion Channel Screening
Andrea Brüggemann; Cecilia Farre, Sonja Stölzle, Claudia Haarmann, Ali Haythorntwaite, Michael George, Niels Fertig, Nanion Technologies, Germany

The importance of ion channels as drug targets, as well as for drug safety issues (e.g. hERG), is widely recognized in industry and academia alike. In recent years electrophysiological screening platforms have been developed to address the need for higher-quality data in lead optimization, secondary drug screening and drug safety testing. Nanion launched a second-generation platform for automated patch clamp - the Patchliner. The Patchliner is a high quality, higher throughput patch clamp workstation-providing stable (>20 min), giga-seal (>1 GOhm) whole-cell recordings with high success rates (60-80%). The Patchliner has up to 8 recording channels, which make it suitable for safety pharmacology, target validation and academic research. It has been successfully used for screening troublesome (sticky) compounds targeting the hERG channel, showing the expected pharmacology, as will be presented.The chip consumables used with the Patchliner come in a microfluidic cartridge for rapid and controlled delivery of solutions to both the internal (!) and external side of the cell membrane. Solution switching is completed within 50 ms, making the Patchliner ideally suited for studies on fast ligand gated ion channels that require rapid and precise application of drugs. Studies on ligand-gated channels, such as glycine and GABA, will be presented. Also, the design of the chip allows for an unlimited number of applications of drugs and wash-buffers, which is beneficial in experiments requiring many solution applications per analyzed drug concentration. In addition to cell lines the Patchliner is also suitable for primary cells and primary cell cultures. Different Primary cells from patients including synoviocytes, T-lymphoblasts, saphenous vein cells and neutrophils were successfully characterized. The Patchliner is suitable for advanced research applications as well as for routine experiments, such as safety testing, where high-quality data is as essential as increased throughput. The intuitive user interface allows the user to run standard protocols for operation in true stand-alone mode, but is also fully interactive allowing for protocol changes “on-the-fly” during experiments.

1. Fertig N, Blick RH, Beherends JC: Whole cell patch clamp recording performed on a glass chip. Biophysical Journal (2002) 82: 3056-3062. 2. Brüggemann A, Soelzle S, George M, et al.: Microchip technology for automated and parallel patch-clamp recording. Small (2006) 2: 840-846. 3. Farre C, Stoelzle S, Haarmann C et al. Automated ion channel screening: patch clamping made easy. Expert Opinion Therapeuthic Targets (2007) 11(4) 557-565.

QPatch: Taking Electrophysiology Further
Morten Rytter Sunesen, Sophion, Denmark

QPatch HT represents the only available Automated Patch Clamp (APC) instrument running 48 giga-seal based whole cell recordings in parallel. In this presentation the latest developments within both ligand and voltage gated ion channel applications on QPatch HT will be presented. Special focus will be on throughput and the uncompromised quality of the recordings. At Sophion we believe that running an APC platform covering both high quality and throughput must be equipped with adequate data analyses possibilities. In the presentation, the ease of data extraction on QPatch HT will be demonstrated.

Selecting the Best HTS Hits to Move Forward – Ligand Binding Characterization Provides Guidance
Ronan O’Brien and Richard K. Brown, MicroCal, USA

Drug discovery programs often rely on high throughput screening (HTS) to provide initial “hit” compounds. These compounds are subsequently optimized through medicinal chemistry modification. Frequently, multiple hits belonging to different chemical classes or “scaffolds” are identified.

An early challenge in the drug discovery and development process is to prioritize these screening hits according to their likelihood for medicinal chemistry development success. Microcalorimetry, both isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) are being effectively applied and combined with other biophysical measurements such as X-ray crystallography and NMR to provide guidance for hit selection and prioritization.

For example, during secondary screening, ITC provides an effective, and nearly universally applicable, means to determine ligand binding constants that can be correlated with observed biological activity from the HTS screen. From the same experimental data, an indication of the source for binding activity (e.g. hydrogen bonding and van der waals interactions versus hydrophobic interactions) is also provided. When multiple hits with similar Kd’s or similar IC50’s are found, it is often advantageous to start with those compounds with the best hydrogen bonding and van der waals interactions since these attributes can be very difficult for the medicinal chemist to engineer back into a molecule whose binding is otherwise largely due to hydrophobic interactions. Recent advances in ITC instrumentation have significantly reduced the amount of material required and the time required for these measurements. Modern calorimetry provides a native in-solution, label-free and immobilization-free method to identify hits most likely to succeed in lead optimization.

Similarly, DSC is being used to rapidly triage large numbers of HTS hits by determining rank orders of affinities. It is also able to identify the “outliers” which may be of great interest, ones that bind to variant (unfolded) forms of the target being screened. Recent advances in automated DSC instrumentation allow these measurements to be acquired with completely unattended operation.

These techniques are also being applied in fragment screening programs. Fragment screening provides an alternative pathway to HTS for finding early lead compounds. Similar to HTS, however, the calorimetric data provides early insights into the degree of binding affinity and mechanism of binding.

This presentation will review the application of these microcalorimetric techniques and the combination of these techniques with other biophysical methods to provide guidance for hit selection and prioritization.

Minimum-Bias Approaches in Biomolecular Analysis
John Hassard (Imperial College, deltaDOT), Stuart Hassard (deltaDOT), Dimitris Sideris (Imperial College), Gary Taylor (deltaDOT), UK Paul Tjossem (Grinnell College IA, USA)

The Label Free Intrinsic Imaging approach to has potential to transform a wide range of separation sciences, and has been successfully applied in small molecule, biologic, nucleic acid, viral and bacterial areas. We show how simple multipixel analyses can result in huge gains in separation analytical power, and do so in a way, which introduces the fewest biases into these analyses. Application areas covering antibody analysis, viral titre, QC/QA, vaccine development, chiral separations and biomarker discovery will be covered.

Incorporating Transmitted Light Modalities into High-Content Analysis Assays
Robert Graves, GE Healthcare, USA

The use of high-content cell-based assays has increased in both research and pharmaceutical drug discovery, enabled by developments in automated auto-focusing fluorescent microscopes, coupled with fast image analysis software. These plate-based assays are traditionally performed with multiplexed fluorescent probes such cell permeable stains, labeled antibodies or fluorescent reporters like GFP.

Images from such assays are amenable to high-throughput analysis using a variety of software algorithms. The most accurate cell-by-cell detection is usually obtained using an image of fluorescently stained nuclei. Even under conditions of confluency, nuclei typically present the easiest targets for the analysis “de-clumping” of closely spaced cells. However, the fluorescent DNA stains have associated toxicity which limits their use to fixed endpoint assays, or short-duration live-cell assays, and are therefore not suitable for monitoring live cells over a period of several days. Cell compartments such as nuclei can be fluorescently tagged with suitable reporters in stable expression systems, but there is considerable assay development and validation required with such approaches.

One alternative is to incorporate transmitted light imaging and analysis into high-content biology. The IN Cell Analyzer 1000 from GE Healthcare provides full fluorescent imaging capability coupled with transmitted light imaging modalities (bright field, phase contrast and differential interference contrast). Using the advanced image pre-processing features available in the IN Cell Investigator analysis software, it is possible to achieve comparable cell detection from phase contrast images compared to fluorescent images. This study demonstrates the use of transmitted light modalities alone, or in conjunction with fluorescent wavelengths, for applications including cell counting and cell morphology.

The Application of Cell-Based Impedance Technology in Drug Discovery
Yama Abassi, ACEA Biosystems, USA

Cell-based assays are an important part of the drug discovery process allowing for interrogation of targets and pathways in a more physiological setting compared to biochemical assays. One of the main hurdles in cell-based assay field is to design sufficiently robust assays with adequate signal to noise parameters while maintaining the inherent physiology of the pathway or target being investigated. Conventional label and reporter-based cell assays may be more prone to artifacts due to considerable manipulation of the cell either by label or over expression of targets or reporter proteins. Cell-based label-free technologies preclude the need for cellular labeling or over expression of reporter proteins, utilizing the inherent morphological and adhesive characteristics of the cell as a physiologically relevant and quantitative readout for various cellular assays. Furthermore, these technologies utilize non-invasive measurements allowing for time resolution and kinetics in the assay. We will discuss the various impedance-based label free technologies and their main applications such as compound-mediated cytotoxicity and receptor signaling in drug discovery.

The CellKey™ System: A Label-Free Cell-Based Assay Platform for the Full Spectrum of Drug Discovery
Applications Ryan McGuinness, MDS Analytical Technologies, USA

Label free cell based assay technologies are advancing at a rapid rate and promise to enhance compound screening efforts by providing novel readouts in simplified formats. One such label-free platform is the CellKey™ System, an impedance-based assay technology. Using electrical measurements, the system generates functional data which reflect integrated cellular responses to compound treatment in a kinetic and noninvasive manner. By not requiring labels or artificial mediators of any sort, the CellKey™ System monitors the activity of native response pathways, thus producing more biorelevant data. The CellKey™ System enables measurement of endogenous and transfected receptors in both primary and immortalized cells. It is also a universal assay platform in that a wide variety of cellular receptor families can be evaluated simultaneously. In this presentation we will provide an overview of this important label-free technology and discuss its applications to the full spectrum of drug discovery tasks, including target identification and validation, compound screening, and lead optimization. We will present case study data on a variety of cellular receptor targets ranging from GPCR’s to ion channels. Through its unique features and strengths, the CellKey™ System has the potential to identify higher quality validated compounds earlier in the drug discovery process.

Optical Resonators for Label-Free Detection: Photonic Crystals, Microrings, and Lasers
Brian Cunningham, University of Illinois, USA

Since the introduction of surface plasmon resonance (SPR), several types of passive optical resonators have been adapted for label-free biosensing, in which some property of light reflected from or transmitted through a transducer is monitored. A key to obtaining high detection resolution for applications such as small molecule detection or low analyte concentration sensing is to design a sensor that produces sharp resonant features, so that small shifts in resonant wavelength or coupling angle can be easily discriminated. Recently, several passive optical resonator technologies have been introduced with detection resolution that is superior to SPR. These technologies include photonic crystal sheets, silicon waveguide microrings, whispering gallery microspheres, and silicon oxide toroids. In addition, new classes of active optical resonators have recently been introduced that are capable of generating their own light output in the form of wavelength-tuned laser radiation, which offers superior resolution and simple optical instrumentation. This talk will discuss these emerging technologies in terms of their detection sensitivity, ease of use, detection instrumentation complexity, and throughput in light of applications in drug discovery such as single-molecule detection, cell-based assays, microarrays, and high throughput biomolecular screening.

Keynote Presentation
The Evolution of Real-Time Label-Free Biosensor Applications
David Myszka, University of Utah, USA

We have come a long way since the first commercially available biosensor was released in 1990 by the biosensor division of Pharmacia. This technology, called Biacore, sparked a revolution in how we study molecular interactions. For the first time in our planets history it was possible to monitor the reversible associations of molecules in real time without labeling. What was initially developed as an immunosensor has now grown into application areas that were inconceivable to imagine twenty years ago. Testament to this growth is the fact that this year we will likely see the 10,000th publication involving the use of Biacore technology. Further proof that the technology has been accepted and is now embedded deep in our research psyche is the observation that there are currently more than 25 different manufacturers of biosensor technology. And, as new systems emerge they are being engineered more and more for dedicated applications. We are fortunate to be living in the Jurassic period for biosensor technology with no asteroids in sight.

SPR Based Direct Binding Assays in Drug Discovery
Walter Huber, F.Hoffmann-La Roche, Switzerland

During the last decade the drug discovery community experienced a growing focus on direct binding assays. Different detection principles (NMR, ITC, AUC, SPR, acoustic waves etc) have been successfully introduced and many scientifique publications report on their positive impact on drug discovery projects.

SPR based direct binding assays cover presently the broadest field of application when considering applications to different types of molecules but also when considering the lifecycle of pharmaceuticals. Interactions between biological macromolecules as well as between proteins with small synthetic molecules in a range of molecular weigth as small as 150 Da have been investigated by SPR based binding assays. The technique is used in target identification, hit finding and validation, lead optimization and when considering biopharmaceuticals even in immunogenicity testing and quality control.

This presentation focuses exclusively on the use of the technology during the discovery phase of small synthetic molecules. Precautionary measures that have to be taken for a successful application in this field will be introduced in detail. This discussion is followed by case studies of four different pharmaceutical relevant targets. Two examples will describe the use of the technique for the hit finding from a focused and a fragment library, respectively, with emphasis on the need for effective affinity and selectivity filtering routines to select the best molecules for further work.. In addition, the value to determine kinetic data (kon, koff) of binding for the assessment of ligand molecules will be demonstrated. The investigation of structural consequences of the binding of small molecules on biomolecules and their impact on the mechanism of inhibition and the inhibitory potency will be demonstrated with an additional example.

Productive and Promiscuous Hits – Biophysical/Label Free Assays in Hit Discovery and Verification
Johannes Ottl, Novartis, Switzerland

Currently we are systematically establishing biophysics assays at Novartis Research to better understand the mechanisms of binding and activity for our hit lists and lead candidate compounds. The goal is to advance validated hits to generate lead candidate compounds more efficient and to enable a better selection for X-ray, late stage biology, CADD, and chemistry. We have analyzed available technology tool sets for their suitability and we are implementing some of them into our HTS and lead discovery flowchart. Examples from the biophysics evaluation studies and running hit discovery projects will be shown to address common challenges of hit identification, hit verification and lead candidate selection: compound aggregation and insolubility, true and promiscuous protein binder and how they can be identified and distinguished. Technologies discussed will be e.g. Dynamic Light Scattering, Differential Scanning Fluorimetry, Surface Plasmon Resonance, Epic Corning, and Mass Spectrometry. The role of certain technologies in the lead discovery flowchart, their advantages as well as pitfalls and limitations will be highlighted with examples.

Harnessing Optical Label-free on Microtiter Plates for Lead Finding: From Binding to Phenotypes
Julio Martin, GlaxoSmithKline, Spain

Some drug targets turn out to be reluctant to screening owing to the lack of a practical or valid biological assay. Likewise, some screening assays may not be predictable of compound activity in a more disease-relevant scenario, or assay development may demand excessive allocation of resources (i.e. time, money or people) at risk of not knowing the actual tractability of the target. Label-free methodologies have been implemented in microtiter plate format that may fill this gap and complement, simplify or enable practical assays. Optical label-free based on grating resonance have proven to be a versatile platform capable to detect from simple binding events between small compounds and biomolecular targets to complex phenotypic changes in non-engineered cells. We will describe applications where compound collections are screened against different target classes and various assay formats.

Applying Label-Free Technologies to Validate Screening Hits
David Mark, Sr., Roche, USA

Label-Free Direct Binding technology is the method of choice for confirming that screening hits bind directly to the target of interest. A number of such technologies are available, such as, NMR, SPR, Thermo-shift and Biolayer Interferometry. A Hit Validation Workflow will be described that takes advantage of the unique properties of each Label-Free technology to confirm the binding of hits to the target proteins. In particular, we will discuss how we have incorporated the ForteBio Octet Red, a fiber optic based biosensor, into our hit validation workflow.

Lead Identification & Confirmation Using A Label-Free Affinity-Based Screening Platform
David J. Burns, Abbott Laboratories, USA

A number of different high throughput affinity screening platforms have been developed to find small molecules that bind to specific therapeutic targets. At Abbott, a label-free, equilibrium-based affinity screening technology known as ASMS (Affinity Screening using a Mass Spectrometry readout) has been practiced for over a decade.

Case studies describing the strengths and limitations of this platform to find and confirm novel chemical leads will be described along with proof-of-concept studies investigating the robustness of this technique in comparison to other label-free and label-dependent screening approaches.

High-Throughput, Solution-Based Protein Stability Measurements via ThermoFluor® Technology
Matthew Todd, J&J Pharmaceutical Research & Development, LLC, USA

ThermoFluor® technology provides for miniaturized, high-throughput biophysical measurement of protein thermal stability. A dye is frequently included that interacts with non-native protein to yield information as to its folded state. The binding free energy of a ligand increases protein stability thus raising the melting temperature, permitting a direct measurement of ligand affinity, in solution. No biosensor is utilized. At Johnson & Johnson Pharmaceutical Research & Development, L.L.C., its patented ThermoFluor technology is currently exploited not only in HTS applications, but also to triage compound Mode-of Action, to drive Hit to Lead Discovery, and to rank SAR during Lead Optimization. Equally relevant, compounds from HTS campaigns utilizing complimentary technologies are typically profiled via ThermoFluor technology to help elucidate compound Mode-of-Action.

The Pyramid Approach to Fragment-Based Biophysical Screening
Glyn Williams, Astex Therapeutics, Ltd., UK

Astex's Pyramid approach combines X-ray fragment screening with one or more Biophysical methods, applied both in parallel and in series. This has consistently led to the discovery of valuable leads for several difficult targets. The factors which influence the selection of methods and the ways in which they are applied will be discussed, with particular reference to X-Ray crystallography, NMR and calorimetry.

Development of High-Throughput Mass Spectrometric (HTMS) Methods for Underivatized Small Molecules in Biological Samples Spanning in vitro, Cell Culture, and ex-vivo Assays
Can Ozbal2, Tom Holt1, Ming-Juan Luo1, Jun Wang1, Xun Chen1, Bernard Choi1, Maxine Jonas2, William LaMarr2, Qi Luo1, Lorraine Malkowitz1, Yusheng Xiong1, Claude Dufresne1 - 1Merck Research Laboratories, Rahway, NJ - 2BioTrove, Woburn, MA

Drug discovery efforts utilize assays covering a range of formats, from initial in vitro screens through in vivo and ex vivo studies. Label-free techniques are currently preferred as they enable rapid assay development. The detection of a given analyte in biological assays often involves development of multiple methods, each tailored to the specific challenges of the assay format. High-Throughput-Mass Spectrometry (HTMS) is an emerging format for high-throughput screening. Mass spectrometry permits direct detection of analytes without need for derivatization, labeling, or capture technologies, however a coupled separation step is needed to achieve desalting from biological media. Liquid chromatography (LC) has been the preferred separation technique but is somewhat slow, even under UltraPerformance LC® conditions. Label-free LCMS methods offer the advantage of a single analytical tool applicable to a wide range of biological matrices. This broad applicability opens the possibility of using LCMS as a universal quantitative method for a given analyte in a range of assay formats. We used a modification of this approach wherein the separation component LC is replaced by the RapidFire™ high-throughput solid phase extraction (SPE) sampling system. This was successfully implemented to develop an assay spanning in vitro, cell culture, and ex vivo assays. The technique was further elaborated to a two-dimensional HTMS application for biologically relevant small molecules in plasma.

Towards a More Efficient & Faster Lead Optimization
Ernesto Freire, Johns-Hopkins University, USA

Potential lead compounds identified by high throughput screening are usually characterized by micromolar or even weaker affinities towards their intended targets. To become viable drug candidates, their binding affinity needs to be increased by several orders of magnitude while simultaneously improving their selectivity. Traditionally, those goals have been accomplished by performing structure activity relationships (SAR) in which the scaffold under consideration is modified in several ways and the resulting activity is evaluated. Past experience has demonstrated that this is a long and, sometimes, frustrating process. Drug development would benefit greatly if lead optimization could be accelerated. One approach that is proving to be extremely successful involves increasing the dimensionality of SAR by utilizing isothermal titration calorimetry (ITC). By experimentally monitoring not only binding affinity but the thermodynamic forces that determine binding, enthalpy and entropy, it is possible to add two additional dimensions and significantly accelerate the development process. Since the enthalpy and entropy reflect different forces like hydrogen bonding, hydrophobic interactions, etc. they provide a more accurate prescription of the type of interactions (chemical functionalities and the location of those functionalities) that are needed to achieve the optimization goals. In this presentation, several examples will be used to show that a thermodynamic platform allows faster potency and selectivity optimization in conformity with existing rules for the design of drug-like molecules and oral bioavailability.

Integration of Biophysical Methods into the Drug Discovery Process
Matthias Frech, Merck KGaA, Germany

Direct molecular interaction studies are an integral part in our drug discovery process. They are involved either in characterizing protein-protein interactions or detecting interactions of proteins with small molecules. They deliver data, which are relevant for our internal Hit to Lead Milestone decisions. During the last years progress have been made in the use of interaction data to efficiently support our drug discovery. We have implemented different technologies to optimize the use of direct interaction studies for our projects. Biophysical methods, which have the highest impact in their combined use, deliver decisive data during the hit to lead process. Here we review the implementation of the molecular interaction studies into the drug discovery. Projects will be presented how the technology contributes at different stages to drug discovery. The current state of implementation and the use of data within projects will be summarized and future steps in the application of the technology will be discussed.

What Drug Binding Properties Should be Captured in Drug Discovery Lead Identification & Optimization Assays?
David Swinney, Roche, USA

Effective drug action requires a bimolecular interaction of the drug with the target that results in communication of the desired pharmacological response to patients. The binding must also be efficiently coupled to physiology in a manner that provides a tolerable therapeutic index. Ideally, drug discovery assays should be able to capture the events that contribute to the bimolecular interaction (affinity) as well as the events important for communicating the desired pharmacological response to the physiological system (nonequilibrium kinetics and conformational changes). In this talk I will discuss the challenges associated with the identification of binding modes and leads suitable for optimization to medicines.

Use of Label Free Detection Technologies in the Hit-to-Lead Process
Frank Stuhmeier, Pfizer, UK

The hit-to-lead process depends on the use of one or several cell based assays for the target of interest, measuring either the binding of the compound to the target, or the functional effect of the binding event on the signaling cascade, or both. These assays use genetically modified CHO or HEK cell lines and are comparatively easy to develop and to run, and the resulting data are often accurate and precise. They have, however, two main disadvantages: (1) It is now generally accepted that the signaling mechanism in genetically modified CHO or HEK cell lines can differ from the signaling in human cells. (2) The quantitative relationship between a specific assay read-out and the overall in vivo response is often not fully understood. Label free technologies are currently not fully established in the mainstream early stage drug discovery process, but can be used for cell line characterization, mode-of-action studies, SAR generation and primary screening. These technologies measure the overall response of a cell to an external stimulus and may become a valuable tool to improve the physiological correctness of assays. Over the last 18 months, Pfizer Sandwich has evaluated several plate based label free assay technologies. In my talk, I will discuss the different label free systems, and I will summarize the results of one of the evaluations. Furthermore, I will discuss the potential benefits of label free technologies, as well their integration into the drug discovery process.

Label-Free Cellular-Based Assays: Relevance to GPCR Drug Discovery
Jeff Jerman, GlaxoSmithKline, UK

The application of label-free detection methodologies to monitoring cellular activity is increasingly emerging and holds vast promise in providing holistic cumulative readouts predictive of in vivo efficacy. Key to drug discovery is the potential for label-free to increase physiological relevance and mechanistic ‘texture’ in early screening data, allowing more informed decision making of compound triage and ultimately reducing drug attrition. The broad applicability of label-free also offers utility in supporting primary, secondary and tertiary follow-up activities. Outputs from preliminary evaluation of both optical- and impedance-based label-free platforms, investigating established and less-tractable/novel G protein-coupled receptor targets will be described. Considerations underpinning throughput vs. relevance as well as complexity of data interpretation will also be discussed.

Nonradioactive Rb+ Efflux Assay Technology for Screening of Ion Channels
Georg Terstappen, Siena Biotech, Italy

Activation of ion channels leads to a movement (flux) of charged molecular species across the cell membrane, which is associated with a concomitant transient change in membrane potential. Patch-clamp electrophysiology is the most precise and information-rich method for measurement of ion flux (current) and considered to be the ‘gold standard’. In addition, radioactive isotopes of ions that pass through the channel under study can be used as tracers for the configuration of flux assays. Such tracers can be radioactive isotopes of the naturally conducting ion species as in the case of 22Na+ and 45Ca2+ or other ion species, which are conducted by the channel such as 86Rb+ in the case of K+ and nonselective cation channels. In order to circumvent problems associated with the short-half life and high-energy emission of radioactive 86Rb and concomitant safety and environmental hazards I developed a nonradioactive Rb+ efflux assay for the high-capacity analysis of native and recombinant ion channels which has found widespread application in drug discovery and development. This assay is based on atomic absorption spectroscopy (AAS), a technique traditionally used for the detection of trace elements in environmental, biological and medical samples. In this presentation, the label-free Rb+ efflux assay technology and its various ion channel research applications in the pharmaceutical industry will be described.

A Bona Fide High Throughput Screening Campaign to Identify Agonist Compounds Using the 384 Well Cell-based Label-Free Epic® System from Corning®
Ralph J. Garippa, Rachid Hamid, and Theresa Truitt, Hoffmann-La-Roche, Inc., USA Arron Xu, Jeffrey Scibek, David Randle, Changqing Wang and Lori Romeo, Corning, Inc., USA

Label-free detection systems for cell-based readouts have gained in popularity in recent years due in part to the non-invasive nature of the method and the opportunity to query more than one signaling pathway in a single format. In the present work, we sampled nearly 100,000 small molecule compounds for their ability to stimulate a stably transfected GPCR in a CHOK1 cell background. We successfully obtained a throughput of approximately 28,000 compounds per 8-hour workday by taking a platform-based approach which utilized on-board liquid handling systems.

The same compound library was also run in a more traditional FLIPR-based calcuim flux format and the hit list compared between the two assays. To our knowledge, this study marks the first published report of a bona fide label-free high throughput cell-based screen without the aid of a fluoresecent/luminescent reporter, colorimetric readout, or exogenously added detection reagent.

The Generation and Characterization of Conformation-Dependent G-protein Antibodies
Melanie Jay, GlaxoSmithKline, UK

Monoclonal antibodies have been generated that specifically recognise the GTP-bound form of the G-protein sub-unit, Gai. In a series of studies we demonstrate that these antibodies recognise GTP-bound, and not GDP-bound, members of the Gai. family of G-proteins and show selectivity for this class of G-proteins over other members of this super-family. Furthermore, we demonstrate the use of these antibodies in cellular imaging studies to detect receptor activation in both recombinant and primary cell lines. To further characterise the specificity of these antibodies we investigated the use of the SRU-BIND system to determine the affinity and specificity of these antibodies to different activation states of the G-protein. However we were unable to demonstrate an interaction between the receptor and G-protein in this system leading us to conclude that the G-protein exists in subtly different conformations within the cellular and biochemical environments. The antibodies generated in this study will become valuable tools to study GPCR activity in high content imaging studies and may lead to the development of non-radioactive assays of G-protein function for use in compound screening.