2.HTS.pdf

Chapter 8 High-throughput screening D Cronk diverse drugs, including β-adrenoceptor agonists and antagonists,...

Chapter 8 High-throughput screening D Cronk diverse drugs, including β-adrenoceptor agonists and antagonists, benzodiazepines, angiotensin receptor antag- INTRODUCTION: A HISTORICAL AND onists and ultimately monoclonal antibodies. FUTURE PERSPECTIVE Today’s marketed drugs are believed to target a range of human biomolecules (see Chapters 6 and 7), ranging Systematic drug research began about 100 years ago, when from various enzymes and transporters to G-protein- chemistry had reached a degree of maturity that allowed coupled receptors (GPCRs) and ion channels. At present its principles and methods to be applied to problems the GPCRs are the predominant target family, and more outside the field, and when pharmacology had in turn than 800 of these biomolecules have been identified become a well-defined scientific discipline. A key step in the human genome (Kroeze et al., 2003). However, it was the introduction of the concept of selective affinity is predicted that less than half are druggable and in reality through the postulation of ‘chemoreceptors’ by Paul proteases and kinases may offer greater potential as targets Ehrlich. He was the first to argue that differences in chem- for pharmaceutical products (Russ and Lampel, 2005). oreceptors between species may be exploited therapeuti- Although the target portfolio of a pharmaceutical company cally. This was also the birth of chemotherapy. In 1907, can change from time to time, the newly chosen targets Ehrlich identified compound number 606, Salvarsan are still likely to belong to one of the main therapeutic (diaminodioxy-arsenobenzene) (Ehrlich and Bertheim, target classes. The selection of targets and target families 1912), which was brought to the market in 1910 by (see Chapter 6) plays a pivotal role in determining the Hoechst for the treatment of syphilis, and hailed as a success of today’s lead molecule discovery. miracle drug (Figure 8.1). Over the last 15 years significant technological progress This was the first time extensive pharmaceutical screen- has been achieved in genomic sciences (Chapter 7), high- ing had been used to find drugs. At that time screening throughput medicinal chemistry (Chapter 9), cell-based was based on phenotypic readouts e.g. antimicrobial assays and high-throughput screening. These have led to a effect, a concept which has since led to unprecedented ‘new’ concept in drug discovery whereby targets with ther- therapeutic triumphs in anti-infective and anticancer ther- apeutic potential are incorporated into biochemical or apies, based particularly on natural products. In contrast, cell-based assays which are exposed to large numbers of today’s screening is largely driven by distinct molecular compounds, each representing a given chemical structure targets and relies on biochemical readout. space. Massively parallel screening, called high-throughput In the further course of the 20th century drug research screening (HTS), was first introduced by pharmaceutical became influenced primarily by biochemistry. The domi- companies in the early 1990s and is now employed rou- nant concepts introduced by biochemistry were those of tinely as the most widely applicable technology for iden- enzymes and receptors, which were empirically found to tifying chemistry starting points for drug discovery be drug targets. In 1948 Ahlquist made a crucial, further programmes. step by proposing the existence of two types of adrenocep- Nevertheless, HTS remains just one of a number of pos- tor (α and β) in most organs. The principle of receptor sible lead discovery strategies (see Chapters 6 and 9). In classification has been the basis for a large number of the best case it can provide an efficient way to obtain © 2012 Elsevier Ltd. 95 Section | 2 | Drug Discovery collections in sufficient quantities and of sufficient quality to file them by electronic systems, and store them in the most appropriate way in compound archives. This resulted in huge collections that range from several hundred thou- sand to a few million compounds. Today’s focus has shifted to the application of defined electronic or physical filters for compound selection before they are assembled into a library for testing. The result is a customized ensem- ble of either newly designed or historic compounds for use in screening, otherwise known as ‘cherry picking’. However, it is often the case that the HTS departments have sufficient infrastructure to enable routine screening of the entire compound collection and it is only where the assay is complex or relatively expensive that the time to create ‘cherry picked’, focused, compound sets is invested (Valler and Green, 2000). In assay development there is a clear trend towards mechanistically driven high-quality assays that capture the relevant biochemistry (e.g. stochiometry, kinetics) or cell biology. Homogeneous assay principles, along with sensi- tive detection technologies, have enabled the miniaturiza- tion of assay formats producing a concomitant reduction of reagent usage and cost per data point. With this evolu- tion of HTS formats it is becoming increasingly common to gain more than one set of information from the same assay well either through multiparametric analysis or mul- tiplexing, e.g. cellular function response and toxicity (Beske and Goldbard, 2002; Hanson, 2006; Hallis et al., 2007). The drive for information rich data from HTS cam- Fig. 8.1 In France, where Salvarsan was called ‘Formule paigns is no more evident than through the use of imaging 606’, true miracles were expected from the new therapy. technology to enable subcellular resolution, a methodol- ogy broadly termed high content screening (HCS). HCS assay platforms facilitate the study of intracellular phar- macology through spatiotemporal resolution, and the quantification of signalling and regulatory pathways. Such useful data on the biological activity of large numbers of techniques increasingly use cells that are more phenotypi- test samples by using high-quality assays and high-quality cally representative of disease states, so called disease- chemical compounds. Today’s lead discovery departments relevant cell lines (Clemons, 2004), in an effort to add are typically composed of the following units: (1) com- further value to the information provided. pound logistics; (2) assay development and screening Screening departments in large pharmaceutical com­ (which may utilize automation); (3) tool (reagent) pro- panies utilize automated screening platforms, which in the duction; and (4) profiling. Whilst most HTS projects focus early days of HTS were large linear track systems, typically on the use of synthetic molecules typically within a molec- five metres or more in length. The more recent trends have ular weight range of 250–600 Da, some companies are been towards integrated networks of workstation-based interested in exploring natural products and have dedi- instrumentation, typically arranged around the circumfer- cated research departments for this purpose. These groups ence of a static, rotating robotic arm, which offers greater work closely with the HTS groups to curate the natural flexibility and increased efficiency in throughput due to products, which are typically stored as complex mixtures, reduced plate transit times within the automated workcell. and provide the necessary analytical skills to isolate the Typically, the screening unit of a large pharmaceutical single active molecule. company will generate tens of millions of single point Compared with initial volume driven HTS in the 1990s determinations per year, with fully automated data acqui- there is now much more focus on quality-oriented output. sition and processing. Following primary screening, there At first, screening throughput was the main emphasis, but has been an increased need for secondary/complementary it is now only one of many performance indicators. In the screening to confirm the primary results, provide informa- 1990s the primary concern of a company’s compound tion on test compound specificity and selectivity and to logistics group was to collect all its historic compound refine these compounds further. Typical data formats 96  High-throughput screening Chapter |8| include half-maximal concentrations at which a com- pound causes a defined modulatory effect in functional LEAD DISCOVERY AND HIGH- assays, or binding/inhibitory constants. Post-HTS, broader selectivity profiling may be required, for active compounds THROUGHPUT SCREENING against panels of related target families. As HTS technolo- gies are adopted into other related disciplines compound A lead compound is generally defined as a new chemical potency and selectivity are no longer the only parameters entity that could potentially be developed into a new drug to be optimized during hit-finding. With this broader by optimizing its beneficial effects and minimizing its side acceptance of key technologies, harmonization and stand- effects (see Chapter 9 for a more detailed discussion of the ardization of data across disciplines are crucial to facilitate criteria). HTS is currently the main approach for the iden- analysis and mining of the data. Important information tification of lead compounds, i.e. large numbers of com- such as compound purity and its associated physico- pounds (the ‘compound library’) are usually tested in a chemical properties such as solubility can be derived very random approach for their biological activity against a quickly on relatively large numbers of compounds and disease-relevant target. However, there are other tech- thus help prioritize compounds for progression based on niques in place for lead discovery that are complementary overall suitability, not just potency (Fligge and Schuler, to HTS. 2006). These quality criteria, and quality assessment at all Besides the conventional literature search (identifica- key points in the discovery process, are crucial. Late-stage tion of compounds already described for the desired activ- attrition of drug candidates, particularly in development ity), structure-based virtual screening is a frequently and beyond, is extremely expensive and such failures must applied technique (Ghosh et al., 2006; Waszkowycz, be kept to a minimum. This is typically done by an exten- 2008). Molecular recognition events are simulated by sive assessment of chemical integrity, synthetic accessibil- computational techniques based on knowledge of the ity, functional properties, structure–activity relationship molecular target, thereby allowing very large ‘virtual’ com- (SAR) and biophysicochemical properties, and related pound libraries (greater than 4 million compounds) to absorption, distribution, metabolism and excretion be screened in silico and, by applying this information, (ADME) characteristics, as discussed further in Chapters 9 pharmacophore models can be developed. These allow and 10. the identification of potential leads in silico, without In summary, significant technological progress has been experimental screening and the subsequent construction made over the last 15 years in HTS. Major concepts such of smaller sets of compounds (‘focused libraries’) for as miniaturization and parallelization have been intro- testing against a specific target or family of targets (Stahura duced in almost all areas and steps of the lead discovery et al., 2002; Muegge and Oloff, 2006). Similarly, X-ray process. This, in turn, has led to a great increase in screen- analysis of the target can be applied to guide the de novo ing capacity, significant savings in compound or reagent synthesis and design of bioactive molecules. In the absence consumption, and, ultimately, improved cost-effectiveness. of computational models, very low-molecular-weight More recently, stringent quality assessment in library man- compounds (typically 150–300 Da, so-called fragments), agement and assay development, along with consistent may be screened using biophysical methods to detect low- data formats in automated screening, has led to much affinity interactions. The use of protein crystallography higher-quality screening outcomes. The perception of HTS and X-ray diffraction techniques allows elucidation of the has also changed significantly in the past decade and is binding mode of these fragments and these can be used now recognized as a multidisciplinary science, encom­ as a starting point for developing higher affinity leads by passing biological sciences, engineering and information assemblies of the functional components of the fragments technology. HTS departments generate huge amounts of (Rees et al., 2004; Hartshorn et al., 2005; Congreve et al., data that can be used together with computational chem- 2008). istry tools to drive compound structure–activity relation- Typically, in HTS, large compound libraries are screened ships and aid selection of focused compound sets for (‘primary’ screen) and numerous bioactive compounds further testing from larger compound libraries. Where (‘primary hits’ or ‘positives’) are identified. These com- information rich assays are used complex analysis algo- pounds are taken through successive rounds of further rithms may be required to ensure the relevant data are screening (’secondary’ screens) to confirm their activity, extracted. Various statistical, informatics and filtering potency and where possible gain an early measure of spe- methods have recently been introduced to foster the inte- cificity for the target of interest. A typical HTS activity gration of experimental and in silico screening, and so cascade is shown in Figure 8.2 resulting in the identifica- maximize the output in lead discovery. As a result, lead- tion of hits, usually with multiple members of a similar finding activities continue to benefit greatly from a more chemical core or chemical series. These hits then enter into unified and knowledge-based approach to biological the ‘hit-to-lead’ process during which medicinal chemistry screening, in addition to the many technical advances teams synthesize specific compounds or small arrays of towards even higher-throughput screening. compounds for testing to develop an understanding of the 97 Section | 2 | Drug Discovery Assay development Validate assay conditions and undertake assay optimization for screening if required Validate pharmacology and demonstrate assay robustness for screening, usually including testing of a limited number of compounds in duplicate on separate days Compound selection and plating Selection of compounds for screening and preparation of screen ready plates Primary screening Screening of all selected compounds at a single, fixed concentration as n = 1 Selection of ‘hit compounds’ for further testing (typically 1% of compounds progressed) Hit confirmation Screening of ‘hit compounds’ at a single fixed concentration in duplicate against selected target and a suitable control assay to eliminate false positives Selection of ‘confirmed hit compounds’ Potency determination Test ‘confirmed hit compounds’ over a concentration range to determine potency against selected target and in a suitable control assay Fig. 8.2 The typical high throughput screening process. Data variability band Data variability band Separation band Frequency 3 σs 3 σc Low controls Assay signal High controls Fig. 8.3 Illustration of data variability and the signal window, given by the separation band between high and low controls. Adapted, with permission, from Zhang et al., 1999. structure–activity relationship (SAR) of the underlying improved by medicinal chemistry in a ‘lead optimization’ chemical series. The result of the hit-to-lead phase is a process (Figure 8.3). Often the HTS group will provide group of compounds (the lead series) which has appropri- support for these hit-to-lead and lead optimization stages ate drug-like properties such as specificity, pharmacokinet- through ongoing provision of reagents, provision of assay ics or bioavailability. These properties can then be further expertise or execution of the assays themselves. 98  High-throughput screening Chapter |8| Assay development and validation Once a decision on the principal format and readout technology is taken, the assay has to be validated for its The target validation process (see Chapters 6 and 7) estab- sensitivity and robustness. Biochemical parameters, rea- lishes the relevance of a target in a certain disease pathway. gents and screening hardware (e.g. detectors, microtitre In the next step an assay has to be developed, allowing the plates) must be optimized. To give a practical example, in quantification of the interaction of molecules with the a typical screen designed for inhibitors of protease activity, chosen target. This interaction can be inhibition, stimula- test compounds are mixed together with the enzyme and tion, or simply binding. There are numerous different finally substrate is added. The substrate consists of a cleav- assay technologies available, and the choice for a specific able peptide linked to a fluorescent label, and the reaction assay type will always be determined by factors such as is quantified by measuring the change in fluoresecence type of target, the required sensitivity, robustness, ease of intensity that accompanies the enzymic cleavage. In the automation and cost. Assays can be carried out in different process of validation, the best available labelled substrate formats based on 96-, 384-, or 1536-well microtitre plates. (natural or synthetic) must be selected, the reaction condi- The format to be applied depends on various parameters, tions optimized (for example reaction time, buffers and e.g. readout, desired throughput, or existing hardware in temperature), enzyme kinetic measurements performed to liquid handling and signal detection with 384- (either identify the linear range, and the response of the assay standard volume or low volume) and 1536-well formats to known inhibitors (if available) tested. Certain types being the most commonly applied. In all cases the homo- of compound or solvent (which in most cases will be geneous type of assay is preferred, as it is quicker, easier dimethylsulfoxide, DMSO) may interfere with the assay to handle and cost-effective, allowing ‘mix and measure’ readout and this has to be checked. The stability of assay operation without any need for further separation steps. reagents is a further important parameter to be determined Next to scientific criteria, cost is a key factor in assay during assay validation, as some assay formats require a development. The choice of format has a significant effect long incubation time. on the total cost per data point: the use of 384-well low- At this point other aspects of screening logistics have to volume microtitre plates instead of a 96-well plate format be considered. If the enzyme is not available commercially results in a significant reduction of the reaction volume it has to be produced in-house by process development, (see Table 8.1). This reduction correlates directly with and batch-to-batch reproducibility and timely delivery reagent costs per well. The size of a typical screening have to be ensured. With cell-based screens it must be library is between 500 000 and 1 million compounds. guaranteed that the cell production facility is able to Detection reagent costs per well can easily vary between deliver sufficient quantities of consistently functioning, US$0.05 and more than U$0.5 per data point, depending physiologically intact cells during the whole screening on the type and format of the assay. Therefore, screening campaign and that there is no degradation of signal or loss an assay with a 500 000 compound library may cost either of protein expression from the cells with extended periods US$25 000 or US$250 000, depending on the selected of subculture. assay design – a significant difference! It should also be The principal goal of developing HTS assays is the fast borne in mind that these costs are representative for rea- and reliable identification of active compounds (‘posi- gents only and the cost of consumables (assay plates and tives’ or ‘hits’) from chemical libraries. Most HTS pro- disposable liquid handling tips) may be an additional grammes test compounds at only one concentration. In consideration. Whilst the consumables costs are higher for most instances this approximates to a final test concentra- the higher density formats, the saving in reagent costs and tion in the assay of 10 micromolar. This may be adjusted increased throughput associated with miniaturization depending on the nature of the target but in all cases must usually result in assays being run in the highest density be within the bounds of the solvent tolerance of the assay format the HTS department has available. determined earlier in the development process. In order to identify hits with confidence, only small variations in signal measurements can be tolerated. The statistical parameters used to determine the suitability of assays for HTS are the calculation of standard deviations, the coef- Table 8.1 Reaction volumes in microtitre plates ficient of variation (CV), signal-to-noise (S/N) ratio or signal-to-background (S/B) ratio. The inherent problem Plate format Typical assay volume with using these last two is that neither takes into account 96 100–200 µL the dynamic range of the signal (i.e. the difference between the background (low control) and the maximum (high 384 25–50 µL control) signal), or the variability in the sample and refer- 384 low volume 5–20 µL ence control measurements. A more reliable assessment of 1536 2–10 µL assay quality is achieved by the Z’-factor equation (Zhang et al., 1999): 99 Section | 2 | Drug Discovery (3(SD of High Control) + 3(SD of Low Control)) present in the compound wells, and assuming a low Z’ = 1 − number of active compounds, the Z-value is usually lower [Mean of High Control − Mean of Low Control] than the Z’-value. where SD = standard deviation and the maximum possible Whilst there are been several alternatives of Zhang’s value of Z is 1. For biochemical assays a value greater than proposal for assessing assay robustness, such as power 0.5 represents a good assay whereas a value less than 0.5 analysis (Sui and Wu, 2007), the simplicity of the equation is generally unsatisfactory for HTS. A lower Z’ threshold of still make the Z’-value the primary assessment of assay 0.4 is usually considered acceptable for cell-based assays. suitability for HTS. This equation takes into account that the quality of The Assay Guidance Website hosted by the National an assay is reflected in the variability of the high and low Institutes of Health Center for Translational Therapeutics controls, and the separation band between them (Figure (NCTT) (http://assay.nih.gov/assay/index.php/Table_of_ 8.3). Z’-factors are obtained by measuring plates contain- Contents) provides comprehensive guidance of factors to ing 50% low controls (in the protease example: assay plus consider for a wide range of assay formats. reference inhibitor, minimum signal to be measured) and 50% high controls (assay without inhibitor; maximum signal to be measured). In addition, inter- and intra-plate Biochemical and cell-based assays coefficients of variation (CV) are determined to check for There is a wide range of assays formats that can be deployed systematic sources of variation. All measurements are nor- in the drug discovery arena (Hemmilä and Hurskainen, mally made in triplicate. Once an assay has passed these 2002), although they broadly fall into two categories: bio- quality criteria it can be transferred to the robotic screen- chemical and cell-based. ing laboratory. A reduced number of control wells can be Biochemical assays (Figure 8.4) involve the use of cell- employed to monitor Z’-values when the assay is pro- free in-vitro systems to model the biochemistry of a subset gressed to HTS mode, usually 16 high- and 16 low-controls of cellular processes. The assay systems vary from simple on a 384-well plate, with the removal of no more than two interactions, such as enzyme/substrate reactions, receptor outlying controls to achieve an acceptable Z’-value. The binding or protein–protein interactions, to more complex parameter can be further modified to calculate the Z-value, models such as in-vitro transcription systems. In contrast whereby the average signal and standard deviation of test to cell-based assays, biochemical assays give direct infor- compound wells are compared to the high-control wells mation regarding the nature of the molecular interaction (Zhang et al., 1999). Due to the variability that will be (e.g. kinetic data) and tend to have increased solvent Biochemical assays Homogeneous Heterogeneous Alternative Radioactive Non-radioactive Radioactive Non-radioactive Scintillation Fluorescence Filtration ELISA Capillary electrophoresis Absorption DELFIA Frontal affinity chromatography Flash plate Intensity Precipitation Biophysical (NMR, SPR) Bead FRET Radioimmunoassay Quantitative-PCR TRF FP FC Alpha-screen Absorbance (Chemi) Luminescence Fig. 8.4 Types of biochemical assay. 100  High-throughput screening Chapter |8| tolerance compared to cellular assays, thereby permitting Cell-based assays frequently lead to higher hit rates, the use of higher compound screening concentration if because of non-specific and ‘off-target’ effects of test com- required. However, biochemical assays lack the cellular pounds that affect the readout. Primary hits therefore context, and are insensitive to properties such as mem- need to be assessed by means of secondary assays such as brane permeability, which determine the effects of com- non- or control-transfected cells in order to determine the pounds on intact cells. mechanism of the effect (Moore and Rees, 2001). Unlike biochemical assays, cell-based assays (Figure 8.5) Although cell-based assays are generally more time- mimic more closely the in-vivo situation and can be consuming than cell-free assays to set up and run in adapted for targets that are unsuitable for screening in high-throughput mode, there are many situations in which biochemical assays, such as those involving signal trans- they are needed. For example, assays involving G-protein duction pathways, membrane transport, cell division, coupled receptors (GPCRs), membrane transporters and cytotoxicity or antibacterial actions. Parameters measured ion channels generally require intact cells if the functional- in cell-based assays range from growth, transcriptional ity of the test compound is to be understood, or at least activity, changes in cell metabolism or morphology, to membranes prepared from intact cells for determining changes in the level of an intracellular messenger such as compound binding. In other cases, the production of bio- cAMP, intracellular calcium concentration and changes in chemical targets such as enzymes in sufficient quantities membrane potential for ion channels (Moore and Rees, for screening may be difficult or costly compared to cell- 2001). Importantly, cell-based assays are able to distin- based assays directed at the same targets. The main pros guish between receptor antagonists, agonists, inverse ago- and cons of cell-based assays are summarized in Table 8.2. nists and allosteric modulators which cannot be done by measuring binding affinity in a biochemical assay. Many cell-based assays have quite complex protocols, for example removing cell culture media, washing cells, Table 8.2 Advantages and disadvantages of adding compounds to be tested, prolonged incubation at cell-based assays 37°C, and, finally, reading the cellular response. There- fore, screening with cell-based assays requires a sophisti- Advantages Disadvantages cated infrastructure in the screening laboratory (including Cytotoxic compounds can be Require high-capacity cell cultivation facilities, and robotic systems equipped to detected and eliminated at cell culture facilities maintain physiological conditions during the assay proce- the outset and more challenging dure) and the throughput is generally lower. to fully automate In receptor studies, agonists Often require specially can be distinguished from engineered cell lines antagonists and/or careful selection Cell-based assays of control cells Detection of allosteric Reagent provision and modulators control of variability of reagent batches Homogeneous Heterogeneous Binding and different Cells liable to become ELISA functional readouts can be detached from support Alpha-LISA used in parallel – high information content Non-radioactive Radioactive Phenotypic readouts are High rate of false enabling when the molecular positives due to Biomarkers Filtration target is unknown (e.g. to non-specific effects of Reporter gene Radioimmunoassay detect compounds that affect test compounds on cell High content screening cell division, growth, function Yeast 2-hybrid differentiation or metabolism) Growth and proliferation More disease relevant than Assay variability can Second messenger (e.g.cAMP, Ca2+) biochemical asays make assays more Label free technology difficult to miniaturize High throughput electrophysiology Membrane potential No requirement for protein Assay conditions (e.g. production/scale up use of solvents, pH) limited by cell viability Fig. 8.5 Types of cell-based assay. 101 Section | 2 | Drug Discovery Assay readout and detection the radioligand to the target brings it into close proximity to the scintillant, resulting in light emission, which can be Ligand binding assays quantified. Free radioactive ligand is too distant from the Assays to determine direct interaction of the test com- scintillant and no excitation takes place. Isotopes such as pound with the target of interest through the use of radio­ 3 H or 125I are typically used, as they produce low-energy labelled compounds are sensitive and robust and are particles that are absorbed over short distances (Cook, widely used for ligand-binding assays. The assay is based 1996). Test compounds that bind to the target compete on measuring the ability of the test compound to inhibit with the radioligand, and thus reduce the signal. the binding of a radiolabelled ligand to the target, and With bead technology (Figure 8.8A), polymer beads of requires that the assay can distinguish between bound and ~5 µm diameter are coated with antibodies, streptavidin, free forms of the radioligand. This can be done by physical receptors or enzymes to which the radioligand can bind separation of bound from unbound ligand (heterogeneous (Bosworth and Towers, 1989; Beveridge et al., 2000). format) by filtration, adsorption or centrifugation. The Ninety-six- or 384-well plates can be used. The emission need for several washing steps makes it unsuitable for fully wavelength of the scintillant is in the range of 420 nm and automated HTS, and generates large volumes of radioactive is subject to limitations in the sensitivity due to both waste, raising safety and cost concerns over storage and colour quench by yellow test compounds, and the variable disposal. Such assays are mainly restricted to 96-well efficiency of scintillation counting, due to sedimentation format due to limitations of available multiwell filter plates of the beads. The homogeneous platforms are also still and achieving consistent filtration when using higher subject to limitations in throughput associated with the density formats. Filtration systems do provide the advan- detection technology via multiple photomultiplier tube- tage that they allow accurate determination of maximal based detection instruments, with a 384-well plate taking binding levels and ligand affinities at sufficient throughput in the order of 15 minutes to read. for support of hit-to-lead and lead optimization activities. The drive for increased throughput for radioactive assays In the HTS arena, filtration assays have been superseded led to development of scinitillants, containing europium by homogeneous formats for radioactive assays. These have yttrium oxide or europium polystyrene, contained in reduced overall reaction volume and eliminate the need beads or multiwell plates with an emission wavlength for separation steps, largely eliminating the problem of shifted towards the red end of the visible light spectrum waste disposal and provide increased throughput. (~560 nm) and suited to detection on charge-coupled The majority of homogenous radioactive assay types are device (CCD) cameras (Ramm, 1999). The two most based on the scintillation proximity principle. This relies widely adopted instruments in this area are LEADseeker™ on the excitation of a scintillant incorporated in a matrix, (GE Healthcare) and Viewlux™ (Perkin Elmer), using in the form of either microbeads (’SPA’) or microplates quantitative imaging to scan the whole plate, resulting in (Flashplates™, Perkin Elmer Life and Analytical Sciences) a higher throughput and increased sensitivity. Imaging (Sittampalam et al., 1997), to the surface of which the instruments provide a read time typically in the order target molecule is also attached (Figure 8.6). Binding of of a few minutes or less for the whole plate irrespective of density, representing a significant improvement in throughput, along with increased sensitivity. The problem of compound colour quench effect remains, although blue Scintillant compounds now provide false hits rather than yellow. As impregnated bead CCD detection is independent of plate density, the use of imaging based radioactive assays has been adopted widely in HTS and adapted to 1536-well format and higher (see Bays et al., 2009, for example). In the microplate form of scintillation proximity assays the target protein (e.g. an antibody or receptor) is coated on to the floor of a plate well to which the radioligand and test compounds are added. The bound radioligand causes a microplate surface scintillation effect (Brown et al., 1997). FlashPlate™ has been used in the investiga- tion of protein–protein (e.g. radioimmunoassay) and Free radioligand – Bound radioligand – receptor–ligand (i.e. radioreceptor assay) interactions energy absorbed by energy absorbed by (Birzin and Rohrer, 2002), and in enzymatic (e.g. kinase) medium. NO LIGHT bead. LIGHT assays (Braunwaler et al., 1996). Due to the level of sensitivity provided by radioactive Fig. 8.6 Principle of scintillation proximity assays. assays they are still widely adopted within the HTS setting. Reproduced with kind permission of GE Healthcare. However, environmental, safety and local legislative 102  High-throughput screening Chapter |8| considerations have led to the necessary development of absorbed by quenc alternative formats, in particular those utilizing fluorescent- ergy her ligands (Lee et al., 2008; Leopoldo et al., 2009). Through En careful placement of a suitable fluorophore in the ligand via a suitable linker, the advantages of radioligand binding Fluorophore Quencher assays in terms of sensitivity can be realized without the obvious drawbacks associated with the use of radioiso- topes. The use of fluorescence-based technologies is dis- FRET peptide cussed in more detail in the following section. Fluorescence technologies Protease The application of fluorescence technologies is wide- spread, covering multiple formats (Gribbon and Sewing, 2003) and yet in the simplest form involves excitation of Fluorophore Quencher a sample with light at one wavelength and measurement of the emission at a different wavelength. The difference between the absorbed wavelength and the emitted wave- + length is called the Stokes shift, the magnitude of which depends on how much energy is lost in the fluorescence Fig. 8.7 Protease assay based on FRET. The donor process (Lakowicz, 1999). A large Stokes shift is advanta- fluorescence is quenched by the neighbouring acceptor geous as it reduces optical crosstalk between photons from molecule. Cleavage of the substrate separates them, the excitation light and emitted photons. allowing fluorescent emission by the donor molecule. Fluorescence techniques currently applied for HTS can be grouped into six major categories: Fluorescence intensity or a quenching group; this results in measurable photon Fluorescence resonance energy transfer emission by the acceptor. In simple terms, the amount of Time-resolved fluorescence energy transfer from donor to acceptor depends on the Fluorescence polarization fluorescent lifetime of the donor, the spatial distance Fluorescence correlation between donor and acceptor (10–100 Å), and the dipole AlphaScreen™ (amplified luminescence proximity orientation between donor and acceptor. The transfer effi- homogeneous assay). ciency for a given pair of fluorophores can be calculated using the equation of Förster (Clegg, 1995). Fluorescence intensity Usually the emission wavelengths of donor and accep- In fluorescence intensity assays, the change of total light tor are different, and FRET can be determined either by the output is monitored and used to quantify a biochemical quenching of the donor fluorescence by the acceptor (as reaction or binding event. This type of readout is fre- shown in Figure 8.7) or by the fluorescence of the acceptor quently used in enzymatic assays (e.g. proteases, lipases). itself. Typical applications are for protease assays based on There are two variants: fluorogenic assays and fluorescence quenching of the uncleaved substrate, although FRET has quench assays. In the former type the reactants are not also been applied for detecting changes in membrane fluorescent, but the reaction products are, and their forma- potential in cell-based assays for ion channels (Gonzalez tion can be monitored by an increase in fluorescence and Maher, 2002). With simple FRET techniques interfer- intensity. ence from background fluorescence is often a problem, In fluorescence quench assays a fluorescent group is which is largely overcome by the use of time-resolved fluo- covalently linked to a substrate. In this state, its fluores- rescence techniques, described below. cence is quenched. Upon cleavage, the fluorescent group is released, producing an increase in fluorescence intensity Time resolved fluorescence (TRF) (Haugland, 2002). TRF techniques (Comley, 2006) use lanthanide chelates Fluorescence intensity measurements are easy to run (samarium, europium, terbium and dysprosium) that and cheap. However, they are sensitive to fluorescent inter- give an intense and long-lived fluorescence emission ference resulting from the colour of test compounds, (>1000 µs). Fluorescence emission is elicited by a pulse of organic fluorophores in assay buffers and even fluores- excitation light and measured after the end of the pulse, cence of the microplate itself (Comley, 2003). by which time short-lived fluorescence has subsided. This makes it possible to eliminate short-lived autofluorescence Fluorescence resonance energy transfer (FRET) and reagent background, and thereby enhance the signal- In this type of assay a donor fluorophore is excited and to-noise ratio. Lanthanides emit fluorescence with a large most of the energy is transferred to an acceptor fluorophore Stokes shift when they coordinate to specific ligands. 103 Section | 2 | Drug Discovery Typically, the complexes are excited by UV light, and emit light of wavelength longer than 500 nm. Donor fluorophore Acceptor fluorophore Europium (Eu3+) chelates have been used in immu- noassays by means of a technology called DELFIA (dissociation-enhanced lanthanide fluoroimmuno assay). DELFIA is a heterogeneous time-resolved fluorometric assay based on dissociative fluorescence enhancement. Cell- + and membrane-based assays are particularly well suited to Analyte the DELFIA system because of its broad detection range and extremely high sensitivity (Valenzano et al., 2000). High sensitivity – to a limit of about 10−17 moles/well – is achieved by applying the dissociative enhancement principle. After separation of the bound from the free label, a reagent is added to the bound label which causes Excitation Emission Emission the weakly fluorescent lanthanide chelate to dissociate and light source 615nm 665nm form a new highly fluorescent chelate inside a protective micelle. Though robust and very sensitive, DELFIA assays are not ideal for HTS, as the process involves several binding, incubation and washing steps. The need for homogeneous (‘mix and measure’) assays led to the development of LANCETM (Perkin Elmer Life Sciences) and HTRF® (Homogeneous Time-Resolved Fluo- rescence; Cisbio). LANCETM, like DELFIA®, is based on chelates of lanthanide ions, but in a homogeneous format. The chelates used in LANCETM can be measured directly Fig. 8.8 HTRF assay type: the binding of a europium-labelled without the need for a dissociation step, however in an ligand (= donor) to the allophycocyanine (APC = acceptor)- aqueous environment the complexed ion can spontane- labelled receptor brings the donor–acceptor pair into close ously dissociate and increase background fluorescence proximity and energy transfer takes place, resulting in (Alpha et al., 1987). fluorescence emission at 665 nm. Reproduced with kind permission of Cisbio. In HTRF® (Figure 8.8) these limitations are overcome by the use of a cryptate molecule, which has a cage-like struc- ture, to protect the central ion (e.g. Eu+) from dissociation. depolarized emission can be used to determine the extent HTRF® uses two separate labels, the donor (Eu)K and the of binding of a labelled ligand (Figure 8.11; Nasir and acceptor APC/XL665 (a modified allophycocyanine from Jolley, 1999). The rotational relaxation speed depends on red algae) and such assays can be adapted for use in plates the size of the molecule, the ambient temperature and the up to 1536-well format. viscosity of the solvent, which usually remain constant In both LANCETM and HTRF®, measurement of the ratio during an assay. of donor and acceptor fluorophore emission can be The method requires a significant difference in size applied to compensate for non-specific quenching of assay between labelled ligand and target, which is a major reagents. As a result, the applications of both technologies restriction to its application (Nosjean et al., 2006) and the are widespread, covering detection of kinase enzyme activ- reliance on a single, non-time resolved fluorescence output ity (Jia et al., 2006), protease activity (Karvinen et al., makes the choice of fluorphore important to minimize 2002), second messengers such as cAMP and inositiol tri- compound interference effects (Turek-Etienne et al., phosphate (InsP3) (Titus et al., 2008; Trinquet et al., 2003). FP-based assays can be used in 96-well up to 1536- 2006) and numerous biomarkers such as interleukin 1β well formats. (IL-1β) and tumour necrosis factor alpha (TNFα) (Achard et al., 2003). Fluorescence correlation methods Although an uncommon technique in most HTS depart- Fluorescence polarization (FP) ments, due the requirement for specific and dedicated When a stationary molecule is excited with plane-polarized instrumentation, this group of fluorescence technologies light it will fluoresce in the same plane. If it is tumbling provide highly sensitive metrics using very low levels of rapidly, in free solution, so that it changes its orientation detection reagents and are very amendable to ultra-high between excitation and emission, the emission signal will throughput screening (uHTS) (Eggeling et al., 2003). The be depolarized. Binding to a larger molecule reduces the most widely applied readout technology, fluorescence cor- mobility of the fluorophore so that the emission signal relation spectroscopy, allows molecular interactions to be remains polarized, and so the ratio of polarized to studied at the single-molecule level in real time. Other 104  High-throughput screening Chapter |8| proprietary technologies such as fluorescence intensity dis- tribution analysis (FIDA and 2-dimensional FIDA (Kask Excitation AlphaScreen© et al., 2000) also fall into this grouping, sharing the 680 nm Emission common theme of the analysis of biomolecules at 1O 2 520–620nm AlphaLISA© extremely low concentrations. In contrast to other fluores- Emission 615nm cence techniques, the parameter of interest is not the emis- sion intensity itself, but rather intensity fluctuations. By confining measurements to a very small detection volume A B (achieved by the use of confocal optics) and low reagent concentrations, the number of molecules monitored is kept small and the statistical fluctuations of the number contributing to the fluorescence signal at any instant become measurable. Analysis of the frequency compo- Alpha donor bead Alpha acceptor bead nents of such fluctuations can be used to obtain informa- tion about the kinetics of binding reactions. Fig. 8.9 Principle of AlphaScreen™ assays. With help of the confocal microscopy technique and Reproduced with kind permission of Perkin Elmer. laser technologies, it has become possible to measure molecular interactions at the single molecule level. Single molecule detection (SMD) technologies provide a number of advantages: significant reduction of signal-to-noise ratio, when excited by light at a wavelength of 680 nm, releases high sensitivity and time-resolution. Furthermore, they singlet oxygen that is absorbed by an acceptor bead, and enable the simultaneous readout of various fluorescence assuming it is in sufficiently close proximity ( 20 000 per day for a FLIPR™) it is suffi- powerful genetic screening technique for measuring the cient for screening of targeted libraries, diverse compound protein–protein and protein–DNA interactions that under- decks up to around 100 000 compounds and the confirma- lie many cellular control mechanisms (Tucker, 2002 and tion of a large number of hits identified in less physiologi- reviewed in Brückner et al., 2009). Widely applied in cell cally relevant platforms. and systems biology to study the binding of transcription factors at the sequence level, it can also be used to screen small molecules for their interference with specific protein– protein and protein–DNA interactions, and has recently been adapted for other types of drug–target interactions (Fields and Song, 1989; Young et al., 1998; Serebriiskii et al., 2001). Conventional in vitro measurements, such as Electrode immunoprecipitation or chromatographic co-precipitation (Regnier, 1987; Phizicky and Fields, 1995), require the interacting proteins in pure form and at high concentra- Extra-cellular tions, and therefore are often of limited use. The yeast two-hybrid system uses two separated peptide domains of transcription factors: a DNA-specific binding Cell part (DNB) and a transcription activation domain (AD). The DNB moiety is coupled to one protein (the ‘bait’), and the AD moiety to another (the ‘prey’). If the prey protein binds to the bait protein, the AD moiety is Small pore High-resistance seal brought into close association with the reporter gene, Antibiotic Intra-cellular which is thereby activated, producing a product (e.g. GAL or LAC, as described above, or an enzyme which allows the yeast to grow in the presence of cycloheximide). The Low-resistance addition of a test compound that blocks the specific Common pathway protein–protein interaction prevents activation of the ground reporter gene. Serebriiskii et al. (2001) describe a project electrode in which lead compounds able to block the activation of a specific N-type voltage-gated Ca2+ channel have been identified with a yeast two-hybrid assay. The bait and prey proteins contained domains of two different channel Fig. 8.11 Planar patch clamp, the underlying principle of subunits which need to associate to form a functional high throughput electrophysiology. channel. Reproduced with kind permission of Molecular Devices. 107 Section | 2 | Drug Discovery Label free detection platforms at a preselected spatial resolution. The spatial resolution is largely defined by the instrument specification and The current drive for the drug discovery process is to move whether it is optical confocal or widefield. Confocal towards as physiologically relevant systems as possible and imaging enables the generation of high-resolution images away from target overexpression in heterologous expres- by sampling from a thin cellular section and rejection of sion systems and, in the case of G-protein coupled recep- out of focus light; thus giving rise to improved signal : noise tors, to avoid the use of promiscuous G-proteins where compared to the more commonly applied epi-fluorescence possible. The downside to this is that endogenous receptor microscopy. There is a powerful advantage in confocal expression levels tend to be lower and therefore more imaging for applications where subcellular localization or sensitive detection methods are required. Also, for the membrane translocation needs to be measured. However, study of targets where the signalling mechanism is for many biological assays, confocal imaging is not ideal unknown, e.g. orphan GPCRs, multiple assay systems e.g. where there are phototoxicity issues or the applica- would need to be developed which would be time con- tions have a need for a larger focal depth. suming and costly. Consequently, the application of assay HCS relies heavily on powerful image pattern recogni- platforms which detect gross cellular responses, usually tion software in order to provide rapid, automated and cell morphology due to actin cytoskeleton remodelling, unbiased assessment of experiments. to physiological stimuli have been developed. These fall The concept of gathering all the necessary information into two broad categories, those that detect changes about a compound at one go has obvious attractions, but in impedance through cellular dielectric spectroscopy the very sophisticated instrumentation and software (Ciambrone et al., 2004), e.g. CellKey™, Molecular Devices produce problems of reliability. Furthermore, the principle Corporation; xCelligence, Roche Diagnostics or the use of of ‘measure everything and sort it out afterwards’ has its optical biosensors (Fang, 2006), e.g. Epic™, Corning Inc; drawbacks: interpretation of such complex datasets often or Octect, Fortebio. The application of these platforms in requires complex algorithms and significant data storage the HTS arena is still in its infancy largely limited by capacity. Whilst the complexity of the analysis may seem throughput and relatively high cost per data point com- daunting, high content screening allows the study of pared to established methods. However, the assay develop- complex signalling events and the use of phenotypic rea- ment time is quite short, a single assay may cover a broad douts in highly disease relevant systems. However, such spectrum of cell signalling events and these methods are analysis is not feasible for large number of compounds considered to be more sensitive than many existing and unless the technology is the only option for screening methods enabling the use of endogenous receptor expres- in most instances HCS is utilized for more detailed study sion and even the use of primary cells in many instances of lead compounds once they have been identified (Haney (Fang et al., 2007; Minor, 2008). et al., 2006). High content screening Biophysical methods in High content screening (HCS) is a further development of cell-based screening in which multiple fluorescence read- high-throughput screening outs are measured simultaneously in intact cells by means Conventional bioassay-based screening remains a main- of imaging techniques. Repetitive scanning provides tem- stream approach for lead discovery. However, during porally and spatially resolved visualization of cellular recent years alternative biophysical methods such as events. HCS is suitable for monitoring such events as nuclear magnetic resonance (NMR) (Hajduk and Burns, nuclear translocation, apoptosis, GPCR activation, recep- 2002), surface plasmon resonance (SPR) (Gopinath, 2010) tor internalization, changes in [Ca2+]i, nitric oxide produc- and X-ray crystallography (Carr and Jhoti, 2002) have been tion, apoptosis, gene expression, neurite outgrowth and developed and/or adapted for drug discovery. Usually in cell viability (Giuliano et al., 1997). assays whose main purpose is the detection of low-affinity The aim is to quantify and correlate drug effects on cel- low-molecular-weight compounds in a different approach lular events or targets by simultaneously measuring mul- to high-throughput screening, namely fragment-based tiple signals from the same cell population, yielding data screening. Hits from HTS usually already have drug-like with a higher content of biological information than is properties, e.g. a molecular weight of ~ 300 Da. During the provided by single-target screens (Liptrot, 2001). following lead optimization synthesis programme an Current instrumentation is based on automated digital increase in molecular weight is very likely, leading to microscopy and flow cytometry in combination with hard poorer drug-like properties with respect to solubility, and software systems for the analysis of data. Within the absorption or clearance. Therefore, it may be more effec- configuration a fluorescence-based laser scanning plate tive to screen small sets of molecular fragments (