High Throughput Screening (HTS) Assays: Uses and Formats

High Throughput Screening (HTS) Assays Uses and FormatsThe increasing demands move upon the pharmaceutical industry to engender a rapid unloosenaround of un sheathd do mediciness is a driving f secondor in the automation of the branches at the initial projection permeateing format of do drugs stripping. This has lead to the development of legion(predicate) lavishly throughput blanket (HTS) stoppages, with the increasing miniaturization of the whole process (1). An explosion in genomic and proteomic studies in recent decades has lead to the gene ration of large numbers of utilitarian protein atoms. The physiological function of these proteins has yet to be elucidated, just to a greater extent could be all important(predicate) future drug natess, much(prenominal) as receptors or enzymes conglomerate in disease pathogenesis (2). These orphan receptors git be studied by steep throughput screens of lessened blood corpuscles, which w tripethorn be likely liga nds. These chemical substances corporation be sourced from existing drugs, pharmaceutical compevery chemical heterogeneous libraries or from indispensable products, much(prenominal)(prenominal)(prenominal) as determines or animals (figure 1 3). Chemical libraries atomic number 18 straight off vast, since the advent of combinative chemistry, which produces newfangled raises by steep throughput regularitys. These commixtures great deal then(prenominal) be assessed for biological drill against a specific target jot (most comm entirely a protein), either as a mixed chemical pool, or in parallel. A positive/ mobile action, or tot, smoke then be further explored.Numerous seek detecting get toats that argon suited for automation necessitate been authentic to detect such receptor ligand and enzyme substrate interactions, to allow the strength drug mite to be further explored. Each seek has expediencys and problems and the most commonly applied techniques b e discussed in this review. As research progresses these processes let special to overcome problems created by the progressive automation and miniaturization of the stoppages. Use of computation to analyse the interactions and ask come forth more than information from them is besides increasing (4). Recent advances in the books suggest that future development of HTS is likely to result in ultra-HTS assay formats, which may be at bottom closed systems such as supply capillaries, or on silicon wafer chips.ReferencesFonseca MH List B (2004) Curr Opin Chem Biol, 8, 319-326.Gilchrist A (2004) well(p) Opin Ther Targets, 8, 495-8.Bleicher KH, Bohm HJ, Muller K, Alanine AI (2003) Nat Rev Drug Disc 2, 369-378.Kato R, Nakano H, Konishi H, Kato K, Koga Y et al. (2005) J Mol Biol, 351, 683-92.Techniques in molecular(a) biology, chemistry and their associated branches ar advancing at a rapid rate. This has enabled the mechanisms at a lower placelying many diseases to be explore d at the molecular level. The ever-increasing sophistication of proteomic and genomic research procedures be producing an explosion in the number of doable drug targets. Until the development of gamy school throughput screening (HTS) assays, the time taken to prise the authorization bio operation and usefulness of melds to act on target mites and travel drugs to act to ameliorate symptoms or even cure or prevent a disease from occurring, was a rate-limiting factor. Since the automation of a number of suitable assays for HTS the foreshorten has led to the number of compounds available for testing against targets becoming the limiting factor. This has jilted the growth of combinatorial chemistry, to such an extent that many consider it to be a branch of chemistry in itself.HTS ass be specify as an automate method of conducting a large number of in vitro assays on a small scale (Patrick 2005). Most commonly, 96-well plates of 0.1ml atomic number 18 use for a number of b ioassays to detect the biological activity of compounds which have the potential to be developed as drugs. These may interact with the target, as a ligand-receptor interaction, or may involve prohibition of an enzyme or interaction with a nucleic acid macromolecule. The chemical reaction produces a detectable output change, which give the gate be detected and/or measured. Thousands of chemicals back easily and quickly be screened this expressive style, and only active compounds taken to the next stage of testing to nonplus out if it has the potential to become marketed as a drug. at that place is increasing closet on drug companies to produce new drugs to keep pace with developments in medical research, as well as an increasingly demanding public and dowery holders. HTS technology is a crucial to meeting these demands, and continues to be developed to produce faster, cheaper and more efficient ways of screening compounds during the initial stages of the drug husking process .ROLE OF HIGH THROUGHPUT test IN DRUG SCREENING molecular biology techniques are allowing us to understand more about the mechanisms of disease, hence providing biomacromolecular targets for potential drugs to interact with. Such targets include receptors, enzymes and nucleic acids and may require inhibition (enzymes) or agonistic/antagonist receptor ligand stick to to produce the desire pharmaceutical effect.In addition, studies development proteomic and genomic techniques are revealing more and more orphan receptors these are proteins (predominately), lipids and nucleic acids (and to nigh extent carbohydrates) that are at one time know to be produced by the body, but their messenger and function is un cognise. Using these as targets against which to screen compounds pull up stakes help to elucidate their function, and more importantly, may turn out to be drug target interaction aims which will be beneficial in disease. As the function of these targets is unknown, there a re no lead compounds that could be utilize as a starting stay for exploration, so HTS is particularly beneficial for screening vast numbers of compounds in the hope that at least one will interact with the mystery story target.The number of potential drugs to be screened is vast pharmaceutical companies have libraries of 0.5-2 cardinal synthetic compounds (King 2002) that have non made it through screening to become marketed drugs. There are similarly commercially available libraries of compounds, such as the Chemical Abstract Service (CAS) registry file, which contains 39 million compound (Abraham 2003). Intermediates in synthetic processes to make a nonher drug should in any case be screened, as they may have the desired pharmaceutical correctties. Isoniazid is an mediate and has now been developed into an effective anti-tuberculosis drug. Existing drugs are also outlay screening, as their biological activity may stretch beyond that for which they are intended. For examp le, cyclosporin A was isolated from soil and had been intended for use as an antibiotic, until its immune-suppressive properties were find and for which it is now sold. The bodys own endogenous chemical messengers, such as morphine, which has similar activity to released endorphins, could also be screened as they may picture a lead compound that butt be modified to enhance activity.Combinatorial synthetic processes can also be apply to generate vast numbers of novel compounds, which is crucial to prevent the availability of new compounds universe a limiting factor in drug development, as HTS is able to screen them so quickly (Carell et al. 1995). It is common for combinatorial synthesis to produce mixtures of compounds, which can be tested as a chemical pool or batch by HTS for biological activity against a specific target macromolecule. This means that thousands of compounds can be screened in a very short time and only pools containing biologically active constituents screen ed further. This usually involves deconvolution processes (such as micromanipulation, recursive deconvolution or sequential release from rosin beads utilize for the synthetic process) to identify exactly which component(s) of the pool is/are active, so that they can be isolated and screened further for drug potential (Wilson-Lingardo et al. 1996). It is now being superseded by production of new compounds in parallel, with a bingle component in each well. Potential drug molecules can also be derived from natural sources, such as plant extracts, but these are less abundant as isolation and purification take time. They are often novel, composite plant molecules and can produce unexpected interactions. An example is artemisinin, an effective antimalarial drug developed from extracts from a Chinese plant it has a elevatedly unstable trioxane ring (Ploypradith 2004).There are so many compounds to be tested against a large range of potential drug targets that high throughput methods a re essential to test the numerable combinations of drug and target interaction to find those that are biologically compatible.HIGH THROUGHPUT SCREENING ASSAYSIn theory any assay that can be performed on the laboratory bench could be automated and scaled up to be utilise for HTS. In practice, however, virtually assays are intrinsically more suitable than early(a)s. High throughput screens require the automation of the entire process. This is best achieved if there are as few steps as feasible in the assay ideally the test should be able to be performed in a single well, with addition of the test sample the last key step. Obviously, it is important that the reaction amongst a target and biologically active compound must be readily detectable. It should ideally be detectable with high sensitivity while the products are still mixed together in the well, rather than needing further steps to disrupt or purify components of the reaction mixture. This can be difficult to achieve with automation and will join on the time taken per test, so becoming less efficient and be effective.Assays used for chemical screening can be carrell- quit or cell-establish (Silverman et al. 1998). Cell-free assays use consequences of relatively pure protein targets, such as receptors or enzyme substrates, which minimizes the number of steps required. It also allows for easy perception of biological activity in the wells of reaction mixture. Cell-based assays have the advantage that they are a closer representation of the normal physiology of the chemical environment inside a cell. Receptor-ligand interactions and enzyme inhibition reactions are more likely to be suggestive of what will happen in vivo (Silverman et al. 1998), especially if ligand-gated ion channels are involved. Cell assays also allow specific processes to be studied and the output can be measured. Indirect effects of small molecule/protein binding which trigger substitute(prenominal) messenger systems, such a s calcium ions or cAMP, can also be observed in their biological context. Cells can be manipulated to stub out target molecules on the coat, so that they are available to bind to novel ligands, which may be tagged for detection. Cell assays can also provide additional information about cytotoxicity and bioavailability of a potential drug. Mammalian cells are expensive and can be difficult to culture in automated HT systems, but yeasts can provide a suitable alternative. Microorganisms such as yeasts are easy to propagate and have been demonstrated to have some homologous chemical processes, or can be easily genetically modified to express human processes accurately (Klein Geary 1997).HTS ASSAYS AND DETECTION FORMATSFluorescenceFluorescence occurs when a fluorophore molecule absorbs a high- free nada photon (often in the ultraviolet range) and emits a lower energy photon, which is typically in the visible range of the spectrum. There are many naturally occurring substances which have this intrinsic property, such as luciferin in fire-flies. There are a number of fluorescence based assays available for use in HTS, to detect whether an interaction has occurred amidst target and potential drug molecule during random screening. Fluorescence assays are generally sensitive, versatile, stable, safe and easy to perform, which gives them a neat advantage in automated systems for HTS. They have the disadvantage that quenchers can be present in the sample which dampen the light emission. There may also be background autofluorescence from free reagents in the reaction mixture (Grepin Pernelle 2000).Many of the assays have developed protocols that take these problems into account.Energy transfer formats uniform time resolved fluorescence (HTRF) uses the ion of the rare earth metal lanthamide (Eu3+ ), bound to crypate as a donor molecule. Following laser excitation (at a wavelength of 337nm), energy is transferred from this complex to an allophyocyanin (APC) acceptor molecule. This results in emission of light of 665nm, over a retentive period (milliseconds), which is recorded in a time resolved bearing so that any background fluorescence from free APC or media is not recorded. Peak emission of light occurs at 620nm for unbound Eu-cryptate, so the ratio of 620665nm emissions can be used to quantify biological complexes in solution (see Figure 1). This technique can be widely applied to screening programs and has already been developed into a 1,536 well plate HTS, with plans to expand this to become ultra HTS (uHTS) and for use in cell-based assays.Figure 1 HRTF schematic explanation(from Grepin Pernelle 2000)Fluorescence resonance energy transfer (FRET) is a slightly different form of detection, using the pattern that excitation energy can be transferred amidst cardinal fluorophore molecules. These can be different shells of green fluorescent protein (GFP) or new(prenominal) bioluminescent molecules, such as luciferase (Hu et al. 2005 ).from Becker et al. 2004Fluorescence polarization (FP)FP can be used as the basis for homogeneous HTS assays for enzymatic and ligand-receptor binding interactions. The principle behind this detection assay is that when polarized light hits a small molecule that is binding to a larger (target) molecule, there will be rotational diffusion of the light beam. This change generate by binding can be detected by measuring the light emitted in external and normal planes of the polarized light. There is no interference from absorptive compounds in complex mixtures, as can occur with other fluorescence based techniques, and FP is quick and easy. Because of this it is used widely in high-throughput screening systems. Kim and colleagues (2004) developed a FP assay for the molecular chaperone Hsp90 (heat-shock protein), which is believed to have a role in cancer. They validated the assay for a high throughput format using molecules known to bind Hsp90, such as geldanamycin. The assay can now be used to screen for novel inhibitors of Hsp90, which may lead to a cancer drug being developed. Stricher and others (2005) have developed a high throughput FP assay for the CD4 binding site of human immunodeficiency virus-1 glcoproteins, such as gp120, which are crucial targets to protect against HIV infection. Their assay used a 384-well plate and CD4M33, a mimic of host cell receptor antigen CD4, found on T helper lymphocytes. roughly studies repoint that FP assay technology can also be developed as part of a HT social organization-activity (SAR) theater. Newman Josiah (2004) showed that FP is sufficiently sensitive to differentiate amidst high-affinity small molecule inhibitors interacting with the target and low-affinity ones, with Src kinase activity as a stumper.FP can also be used in cell-based assays, in society with confocal microscopy (Heilker et al. 2005), as it shows high sensitivity even at snatch volumes of reaction mixture, down to femtolitres. This type of assay can be described as fluorescence intensity dispersal anaylsis (FIDA) and measures the absolute concentration of both bound and unbound ligand, thus providing the information with its own internal control. FIDA has been used to explore ligands which bind to G-protein coupled receptors (GPCR), which are widespread throughout the body and involved in signal transduction for numerous cell processes. They are and so important therapeutic pharmaceutical targets, and can be studied in association with membrane fragments from cells over-expressing GPCR or associated with virus-like particles.Fluorescence correlation coefficient spectroscopy (FCS)FCS uses a two-photon excitation to measure the relative fluorescence of different molecules within a homogeneous mixture, from which the amount of each can be calculated. The technique can be applied to measure the relative amounts of ligand bound versus unbound receptor molecules, or cleaved versus intact enzyme substrates. FCS can be co nducted using minute reaction volumes, less than 1 femtolitre (fl) is adequate for this sensitive, fast assay, which can get interactions of single molecules (Sterrer Henco 1997). FCS can also be used to study ligand-receptor interactions in live cells (Pramanik 2004), which allows reactions to be assessed, and to some extent the properties of the interaction quantified, in their biological context. The use of such live cell assays in a high throughput format will provide a wealth of information not observable in chemical solutions alone.Many applications of FCS are conducted in conjunction with confocal microscopy, which allows interaction kinetics to be examined on a molecular level, by the changes in diffusion patterns of the excitation. Confocal microscopy uses a high numerical aperture lens to sharpen the laser, to provide excitation and produces minimal background excitation, which allows such minute quantities to be studied. It can be used to detect spatial and temporal in teractions in live cells, increasing the amount of information that can be obtained and used for drug development (such as interactions with other cell components or pH effects within the cell Zemanova et al. 2003)The dual-colour cross- correlation spectroscopy method of FCS uses two different, spectrally separated, fluorophore molecules, which are attached to each of, for example, a receptor and possible ligand, or potential substrate to be cleaved. The two colours will be observed to fluoresce together if an interaction occurs, or separately if a substrate has been cleaved and the kinetics of this can then be assessed (Kettling et al. 1998). This can be demonstrated using, a DNA bound that has a red fluorophore molecule attached to one and and a green one to the opposing end. The strand is cleaved by restriction endonuclease enzyme ecoRI, which is detected by spectroscopy as a decrease in the quantity of DNA molecules with fluorescence at both ends. The method was shown to be sui table for this type of enzyme kinetics study, by accurate detection of catalytic activity down to an enzyme concentration of 1 pM (pico molar) and proper description of the reaction by the Michaelis-Menten equation. This method, dual-colour FCCS, therefore has great potential for HTS of enzyme and ligand binding reactions.Biomolecular fluorescence/reportersThere are numerous molecules produced by plants and animals naturally that produce fluorescence, or what is sometimes called bioluminescence. Some of these have been adopted as research tools, such as green fluorescent protein (GFP), which produces green light at 509nm pastime excitation by blue light (Arun et al. 2005). The gene that encodes for this fluorescent protein has been elucidated and is now commonly inserted into the genomes of genetically modified microrganisms and cell lines. It is then expressed under the control of desired promoters, often as a fusion protein. In this way patterns of gene expression can be observed and changes in transcription detected. HTS for new drugs use this technology to detect changes in transcription that occur via secondary messenger systems pursuance receptor-ligand binding in a live cell. For example, Changsen and others have validated a GPF microplate assay, using an acetamidase promoter associated with the gfp gene, to screen for antituberculosis drugs (2003), and found it to be suitable for HTS for novel drugs.GFP reporter technology requires a detection system and most of those described for detecting fluorescence from synthetic fluorophores can also be applied, such as FRET (Zhang 2004), FCS and confocal microscopy.FITC (fluorescein 5- isothiocyanate)FITC can be bound to other molecules as a marker and the fluorescence measured robotically in HTS systems. For example, FITC bound to heparin sulphate (HS) has been used to screen for heparanase inhibitors in a HT assay 384-well microtitre plates are used, which are coated with fibroblast growth factor (FGF). T his captures the FITC-HS, and denominate fragments are only released into the media when cleavage by heparanase has occurred. This is quantitatively measured by robotic detection of the amount of fluorescence in solution. Heparanase is believed to have roles in inflammation, tumour angiogenesis and metastasis, so is an important drug target in the insurement of cancer (Huang et al. 2004).ChemiluminescenceSome assays using chemiluminescence have been adapted to use HT formats. These rely on chemical reactions to produce light emission as a side-product, which can be detected. One such HTS uses coupled reactions involving the enzymes acetylcholinesterase, choline oxidase and horse-radish peroxidase, in 96 and 384-well plate formats, to screen for novel acetylcholinesterase inhibitors to become new drugs to treat Alzheimers Disease (Andreani et al. 2005). light proximity assay (SPA)Scintillation proximity assays are used for quantitatively studying binding reactions. The receptor/tar get is bound to a surface such as a plastic bead. The ligand is labelled with radioactive isotype (typically H3 or I125 ) and emit electrons with a short range of about 10um. A twinkling counter under the surface to which the target is bound detects the ligand only when it is bound. When it is free in solution the media absorbs the electrons and they are not counted. This allows binding interactions to be quantified whilst at equilibrium. Zheng and others (2004) have used SPA as part of a HTS and have identified several novel inositol monophosphatase inhibitors, which may be developed as drugs fro bipolar disorder.Mass spectrometryMass spectrometry is currently a popular option for HTS, as it is sensitive, selective and easily automated. It allows the activity, molecular freight (most drug-like molecules are 150-400 Da), elemental composition and structural features of a test compound to be analysed. This wealth of information is of great use for exploring the molecular interactio ns between target and potential drug compounds. A very high throughput can be achieved using flow injection analysis, which does not require any sample preparation. Solutions of the samples are sprayed, using electrospray or APCI (atmospheric pressure chemical ionization), which ionises the molecules in the sample, prior to analysis by the masses spectrometer. Sometimes tandem mass spectrometers are employed, to glean more structural information and elemental composition. The advantage of these techniques are that as well as being very high precision they can be conducted on the original sample, without the need the separate the compound out from a mixture.For LC-MS (liquid chromatography mass spectrometry), semipreparative HPLC (High Peformance Liquid Chromatography) is often used before the HTS to verify the organise and purity of each compound to be tested, especially those from a combinatorial library. Improving purity in this way facilitates more accurate observation of any biological interactions that occur between the target and the test compound, as well as easing meter reading of structural information about the test molecule or changes induced by the interaction (Abraham 2003). HPLC is easily automated and involves detection of a UV signal above a threshold level, which triggers collection of the fraction. Several fractions may be obtained from one sample, or the ready reckoners controlling sample collection can be programmed to detect only at peaks of desired molecular ions, following ionisation by a suitable technique, such as electrospray or MALDI (matrix-associated laser desorption/ionisation), which can be used as a ennoble way of ionising more fragile molecules (Hillenkamp et al. 1991). LC-MS can be slower than other approaches but is sometimes necessary. Further developments to speed up the automated process include parallel LC-MS, in which multiple HPLC columns are interfaced to a single mass spectrometer (Kenseth Coldiron 2004), and f ast HPLC.NMRNMR is a useful technique for exploring the 3-dimensional anatomical structure of biomacromolecules, in a concentrated solution. It is limited by the small size of molecule amenable to this technique typically at a lower place 30kDa, so is more useful for small drug-like molecules than the molecular target they interact with. Structure-activity relationships (SAR) can be studied by observing alterations in a proteins NMR spectrum. This not only indicates that ligand binding has occurred, but can give an indication of the location of the binding site (Shuker et al. 1996).roentgenogram crystallographyX-ray crystallography enables the 3-dimensional structure of protein molecules to be studied, with resolution to the atomic level. The technique requires the molecule to be studied in its crystalline form, which is not a problem for the volume of biomacromolecules that are drug targets. Protein crystallization technology has also had to adapt to high throughput methods, so as not to become a bottleneck. Some in full automated systems can now produce as many as 2,500 to 140,000 crystallization experiments a day (Kuhn et al. 2002).Studying the three-D structure of the target often produces clues to the type of ligand that will bind, which speeds up the time taken to find lead compounds in drug discovery. An example of this is the development of antiretroviral drugs used to treat AIDS (acquired immunodeficiency syndrome), such as amprenavir (Agenerase), which followed from the study of the structure of the drug target, HIV (human immunodeficiency virus) viral protease. other drug developed from such structure based studies is zanamivir (Relenza) a flu treatment based on the crystal structure of the surface glycoprotein, neuraminidase, which is crucial for viral infectivity (Varghese 1998). This is likely to be an important tool in the fight against an influenza pandemic.In X-ray crystallography, the macromolecular 3-D crystal is bombarded with X-rays , by a rotating-anode X-ray generator or a synchrotron, and the diffraction pattern produced is detected. Multiple measurements of diffracted waves generate much data, which can be analysed using calculations, such as Fourier synthesis and a structure revealed (Blundell et al. 2002). Advances in the structure determination process have aided the resolution of structures, for example, multiple-wavelength anomalous distribution (MAD), in which selenomethionine is incorporated into proteins that are overexpressed by genetically modified micro-organisms, which simulates isomorphous replacement and allows the phases to be calculated (Hendrickson et al. 1990).Low-affinity binding reactions between ligand and target may have important properties and provide leads that would be missed by other HTS methods.Development of high throughput X-ray crystallography, by increased automation at all stages of the procedure, has lead to its growing use in lead discovery as well as its more traditiona l role in lead optimisation (Abola et al. 2000). This enables the technique to screen compound libraries, including those from combinatorial synthesis. Crystallographic screening for novel ligands in this way has already had some success for example, a new class of urokinase inhibitors have been discovered, for treating cancer (Nienaber et al. 2000).Co-crystallization of receptor-ligand complexes allows the interaction between the molecules to be studied and conformational changes in the target, upon binding, to be discovered. This approach is known to be used by several industrial laboratories and has the capacity to compare the interactions of hit ligands in the generation of a lead series. It also decreases the time taken to explore hits, which is a crucial factor for the pharmaceutical industry (Abraham 2003). Another way of achieving crystallized receptor-ligand interactions is to soak the ligand, often as molecular fragments turn in DMSO (dimethyl sulphoxide), into the recept or protein crystal (Nienaber et al. 2000), and observe changes in electron densities indicative of interaction.Structure based drug design in silicoThree-dimensional structures can also be used in figurer modelling programs to call up which ligands might bond/interact with targets or receptors, as an initial stage of drug design. This is truly a high throughput method as figuring power allows the rate at which ligand-receptor interactions can be practical(prenominal)ly screened to be incomparable to even the fastest high throughput methods involving physical experimentation. This is often termed virtual ligand screening (VLS), or in silico screening (Klebe 2000). Perrakis and his colleagues (1999) combined automated protein model building with iterative structure refinement, using ARP (automatic pattern recognition), which has been crucial for structure based drug design (SBDD). Diffraction data is fed directly into the computer program and a protein crystallization model produce d automatically.Various programs have been developed to assess docking of virtual ligands into known target receptor sites and gain ground of their suitability and fit, determined by energy. Some algorithms seem to have some bias towards certain chemical families this can be reduced by using multiple docking algorithms simultaneously (Charifson et al. 1999).Software programmers and chemical modellers must remember to take into account the natural properties of protein molecules, as they are more flexible and accommodating of small changes than the rigidity suggested by traditional computer programs, although some programs now attempt to recreate this (Schapira et al 2000).Optimal results for structure based drug design are likely to be achieved by combining virtual and experimental methods, such as SAR-NMR technology advocated by Shuker, Fesik and colleagues (1996).MicroarraysDNA microarrays have been constructed following the sequencing of the human genome, using cDNA to study tho usands of genes. From this has stemmed the growth of proteomics and protein biochips, as these are the functional molecules encoded by the genome. Protein arrays/biochips comprise of immobilized proteins, which can be used, in the drug development context, to study ligand-receptor interactions (Lueking et al. 2005). Interactions of known pharmaceutical chemicals with proteins can also be explored. For example, Leflunomide (an isoxazole derivative) has been shown to have anti-inflammatory drug properties in vivo. Analysis of protein interactions using an array revealed that it was not only interacting with the venture mitochondrial enzyme, but a number of other proteins in the cell, such as pyruvate kinase (Mangold et al. 1999). As the majority of drug targets are proteins, and many of the drugs themselves proteins too, protein arrays are likely to become more popular, as well as higher throughput. coatingCHOOSING THE BEST HTS ASSAYThe literature reveals numerous modifications and validated systems of all the possible assays that are suitable for adaptation to high throughput screening in drug discovery. Many of the traditional weak points of each assay have therefore been addressed in this way, making critici