Screening Numbers

The combinatorial biology approaches described above differ significantly from other more directed practices for pathway manipulation. The random elements of pathway isolation and gene shuffling in combinatorial biology that allow the merger of rare and unexpected biosynthetic capabilities also, by necessity, generate a large number of clones. To make combinatorial biology a practical method of natural products drug discovery, a screening system that could screen large numbers of recombinant...

Fpr1

For the ER, RU486 for the PR, and Ro 41-5253 for the RARa) potentially because yeast lacks these cofactors 31,42,52 . Therefore in cases where receptors do not function, it may be necessary to coexpress additional factors. Another important feature is that some receptors are covalently modified (i.e., yeast supports the phosphorylation of the glucocorticoid receptor 62 ) and therefore the appropriate post-translational modification enzymes must be expressed to obtain ligand-dependent activation...

Introduction

Drug discovery in the pharmaceutical and biotechnology companies has seen spectacular changes in the last decade mainly due to technological advances in biology, biochemical assays, genomics, proteomics, combinatorial chemistry, miniaturization, automation, computerization, and information technologies. With this technological revolution, drug screening capacities have increased immensely, allowing the pharmaceutical companies to process an increased number of drug targets rapidly by...

Sourcing Robotic Systems

Because robotic laboratory systems can be customized for the user, there are a number of considerations in selecting a vendor for a robotic system for drug screening and suggestions to new users to improve the implementation process. Users have made a number of recommendations to improve the success of robotic systems 15 . When evaluating vendors, it is recommended to visit the vendor's references, see the system, and ask specific questions regarding the vendor's performance. It is also...

Marcel

As the name indicates, robot controllers send the signals to the motors and coordinate the movement of the motors in the robot arm. In order to move accurately and repeat the desired movements time after time, the motors and controller use bidirectional signals to position the axes motors accurately. Servo motors can be of either the AC or DC type, each having some differences in the motor construction and the type of robot controller (both still operate from the standard alternating current...

Functional Expression Of G Proteincoupled Receptors In Yeast

The superfamily of G protein-coupled receptors (GPCRs), characterized by a similarity in structure consisting of seven transmembrane domains, bind a wide variety of ligands that range from small biogenic amines and lipids to large complex proteins 64 . Upon ligand binding a conformational change occurs in the GPCR, leading to activation of heterotrimeric G proteins via a catalytic exchange of GTP for GDP on the a subunit and dissociation of the a subunit from the Py complex 65 . The free a...

Future Developments In Screening

Given the capacity for modern molecular genetic tools and methods to generate recombinant DNA libraries, it was appreciated at an early stage that the efficient practice of combinatorial biology would require the screening of large numbers of clones. For example, a single genomic library from a single donor strain may contain greater than 105 individual clones. In view of such sample numbers, cost, miniaturization, and throughput become critical factors for screening technology development....

Info

The 384-well plate pros and cons. J Biomol Screening 1 63-64, 1996. AJ Kolb, K Neumann. Beyond the 96-well microplate instruments and assay methods for the 384-well format. J Biomol Screening 2 103-109, 1997. P Pasini, M Musiani, C Russo, P Valenti, G Aicardi, JE Crabtree, M Baraldini, A Roda. Chemiluminescence imaging in bioanalysis. J Pharm Biomed Anal 18 555564, 1998. AM Maffia, I Kariv, KR Oldenburg. Miniaturization of a mammalian cell-based assay luciferase reporter...

Types of Robots

There are various types of robots in use in laboratories, some being better suited for a particular application than others. The primary types of robots used in today's laboratories are of the ''articulated arm'' and ''cylindrical coordinate'' style. Articulated arm robots are characterized by an arm with a bending joint in the middle, sometimes referred to as the ''elbow'' joint, with the obvious anthropomorphic reference to the human arm. This type of jointed arm allows the robot to work...

Discovery Techniques Not Suited For

Without question, electrophysiological methods have revolutionized our understanding of the properties and behavior of ion channels and their interactions with drugs 57 . Patch clamp electrophysiology refers to a technique for forming a tight seal on the cell surface with a pipette containing a small electrode. This allows the current flow through this small patch (several m2) to be recorded for analysis of single-channel properties or whole-cell electrical properties. However, traditional...

References

Drug discovery a historical perspective. Science 287 1960-1964, 2000. 2. JR Lakowicz. Principles of Fluorescence Spectroscopy. New York Plenum Press, 1983. 3. AJ Pope, UM Haupts, KJ Moore. Homogeneous fluorescence readouts for miniaturized high-throughput screening theory and practice. Drug Disc Today 4 350-352, 1999. 4. RA Jefferson. Assaying chimeric genes in plants. The GUS gene fusion system. Plant Molec Biol Reporter 5 387-405, 1987. 5. AG Rao, P Flynn. A quantitative assay for...

Bar Code Scanner

Another important peripheral device for sample tracking is a bar code scanner. Since bar code scanners are small, they can be mounted at many different places in the systems. They can be mounted on the robot arm, above the gripper, and, as part of the robot, they can be taken to the plate for scanning at various locations in the robotic system. Alternatively, the scanner can be mounted in a fixed location on the table and plates can be taken to it for identification and then processed according...

Expression of Heterologous Proteins to Get a Phenotype

Expression of Steroid Hormone Receptors in S. cerevisiae Screens to find ligands for steroid hormone receptors such as the retinoid receptors have been designed in S. cerevisiae 32 . Steroid hormone receptors occur intracellularly. They have a ligand-binding domain, a dimerization domain, and a transactivation domain. When ligands induce these receptors to homo- or heterodimerize, their transactivation domain binds the specific response elements and activates specific promoters. The...

Determining Proteinprotein Interaction

Understanding the interactions of a given protein with other proteins and macro-molecules within a cell goes a long way towards understanding the biology of the cell and also towards understanding the role of individual proteins 38-43 . Protein interaction maps are being developed by using the yeast two-hybrid technology and a complete protein interaction map of Saccharomyces cerevisiae has been published 44,45 . In order to obtain a complete analysis, 6000 ORFs were fused to the Gal4...

Assay Systems For Addressing New Targets

Assays can be developed for orphan GPCRs, proteases, kinases, and phosphatases without knowing the cellular function of the new protein. For other classes of proteins, especially proteins with unknown activity, alternative technologies are being developed. Figure 1 summarizes how new gene targets and protein technologies are being coupled with combinatorial chemistry, microarrays for gene expression and in vivo studies to develop drugs. The yeast two-hybrid system is being used extensively to...

Ligandbinding Formats

Binding assays for compounds that interact with ligand-gated ion channels are well tailored for HTS and have been used extensively in discovery programs directed toward GABA-, glutamate-, purine-, serotonin-, inositol-1,4,5-triphos-phate-, and nicotine-responsive channels. In addition, voltage-gated ion channels, although lacking endogenous ligand activation, have been studied using binding paradigms. This has been possible where radio- or fluorescence-labeled drugs or toxins bind to the...

Incubators

Many assays used in drug discovery methods utilize an incubation period at either room temperature or at an elevated temperature such as 37 C. Many assays use live cells that require humidity and a CO2 atmosphere. Such incubators are a key requirement for robotic systems for drug screening. Some robotic systems use commercial incubators that have been modified for robotic use by adding an automatic door opener. But these designs are not optimal for robotic systems, because the single door faces...

Validation Of New Targets

Genetic methods have been applied to the validation of genes as drug targets. Koch's postulates, devised as a set of criteria for determining whether a given microbe is the cause of a particular infectious disease, can also be applied to the validation of new genes as drug targets. In applying these postulates to the field of genomics, if a new gene putatively implicated in disease is isolated, a mutant form of it should result in the disease phenotype, and reintroduction of the normal gene...

Moving Into the Third Millennium After a Century of Screening

For many decades, the discovery of drugs has depended on screening. From the previous century until the 1960s, chemical compounds and natural products were tested in whole animal assays, and in the case of antibacterial and antitumor testing, the samples were tested in whole cell assays. Although screening was labor intensive and expensive, it was fruitful. A large number of successful drugs such as the cephalosporins, aminoglycosides, tetracyclines, macrolide antibiotics, doxyrubicin,...

Elisa

ELISA is frequently run on an automated system because it has many time-consuming steps including the addition of reagents, performing timed incubations, and subsequent plate washings before reading the plate in a plate reader. Most ELISA assays produce a color or fluorescence end product that, when the reaction is stopped, gives an end point reading. A robotic system is an ideal platform to perform ELISA assays because the forward scheduling software assures that the robot will perform the...

Intracellular Receptors

The intracellular hormone receptors, unlike the membrane receptors, are located either in the cytoplasm or in the nucleus of the cell. The nuclear receptor (NR) superfamily is composed of steroid receptors including androgen, estrogen, glucocorticoid, mineralcorticoid and progesterone receptor, nonsteroidal receptors like thyroid hormone, vitamin D and retinoic acid receptors, ecdysone receptors, which are found in insects, and orphan NR receptors (Table 3), which constitute the majority of the...

Encapsulating Other Organisms

The three macrodroplet formats are shown to work effectively with actinomy-cetes. Application of this approach to other ''biosynthetically talented'' organisms such as fungi and myxobacteria is explored. Both are useful sources of chemical diversity and much is known about their genetics. We have done some preliminary combinatorial biology on both types of organisms. Since both types are used to produce natural products through fermentation, the macrodroplet approach is adopted to screen the...

Application Of Human Hepatocytes In The Evaluation Of Drugdrug Interactions

Mechanism and Clinical Significance of Pharmacokinetic Drug-Drug Interactions Multiple drug therapy is widely practiced either to treat a medical disorder or to treat several concurrently existing ailments in the same patient. It is now known that drugs may interact, resulting in serious pharmacological and or toxicological consequences. Drugs interact mainly by a phenomenon known as pharmacokinetic drug-drug interactions alteration of the metabolic clearance of a drug by a coadministered...

Fluorescence Technologies

Since fluorescent molecules can be excited many times, the total number of photons obtained from a sample is simply a function of the quantum yield, the number of fluors present, and the number of excitation photons presented. This effect amplifies the signal and improves the measurement statistics. Sufficiently sensitive detection of fluorescence emission permits even single-molecule detection. Over the years, several fluorescence methods have been developed to address a wide range of...

Advantages of Primary Hepatocytes in Drug Metabolism

Hepatocytes represent a physiologically-relevant experimental model of the liver because of the following properties 1. Physiological Enzyme, Cofactor, and Substrate Concentrations Freshly isolated hepatocytes contain complete, undisrupted enzymes and cofac-tors at the same level as the liver in vivo, therefore allowing the generation of data similar to hepatic metabolism in vivo. 2. Relevant Drug Concentrations Hepatocytes in culture retain active uptake as well as biliary excretion similar to...

M A R C B L

Base access tools consider the records to be the desired output from a database query. In reality, the user often spends a considerable amount of time computing summary statistics and preparing charts of the data. The tools developed for HTS should take the user straight to these summaries and charts to save time. Additionally, the full power of the chemical database needs to be exploited to build structural models to guide follow-up synthetic work. Tremendous gains in the ability to generate...

Case Study of Multidimensional FCS Readout

A novel assay based on a multiparameter fluorescent readout is described below. A theophylline antitheophylline antibody interaction was chosen as a pharmacologically relevant assay system in order to demonstrate the full potential of EVOTEC's multidimensional fluorescence analysis based on its proprietary FCS+ detection platform. Theophylline therapy has been a cornerstone of asthma therapy through the years. Theophylline inhibits the breakdown enzyme phosphodiesterase with a resultant...

Conclusions

Defects in ion channel function clearly underlie a variety of clinically and economically important human diseases. The corresponding importance of ion channels as drug targets is reflected in the heightened attention these proteins have received in drug screening programs in recent years. Unfortunately, electrophysi-ology is the technology of choice for the detailed characterization of ion channel behavior but is not well suited for HTS. Instead, traditional methods that have less sensitivity...

Mj

Immobilization of membranes containing receptor using proprietary coating procedure (96- and 384-well FlashPlate microplates) Incubation with radioligand and compounds Bound radioligand detected in Top Count Figure 21 Schematic representation of FlashPlate technology. Receptor immobilized to a 96- or 384-well FlashPlate is incubated with radioligand and compounds. Receptor bound radioligand is in close proximity with Scintillant and is detected in a TopCount. (Courtesy of Packard, a Packard...

B TRF and HTRF

Separation or wash steps may not be acceptable for miniaturized assays. Ideal assays for miniaturized formats will be in a simple mix-and-read, homogeneous format. Recently developed FRET-based approaches combine the benefits of a highly sensitive fluorescent label and a homogeneous assay protocol under a variety of HTS experimental circumstances. One of these systems, called homogeneous time-resolved fluorescence (HTRF), involves fluorescence resonance energy transfer between a slow-decay...

Small or Large

An even more important reason to propose systems that are specifically developed for an application or for a family of applications is to move away from the custom integration of large and complicated systems. In general, large systems are expensive, not just because of the hardware, but because of the additional amount of system integration time and project management resources that are required. Thus large systems take disproportionately more time to be implemented than small systems. If the...

Ion Channelrelated Diseases

Defects in ion channels are either responsible for or strongly implicated in a growing number of diseases, a reflection of their ubiquitous expression and central role in cellular homeostasis. In some cases, the molecular characterization of an ion channel has led to the discovery of genetic defects in channel structure function. In other cases, genetic investigations of human disease, or more typically mouse and fly mutants, have led to the identification of new members or classes of ion...

Conclusion

Drug screening for identifying novel chemical structures for different targets is moving to HTS and UHTS. The assays have to be simple, homogeneous, robust, reproducible, and fully automatable to adapt to HTS. The design of a cell based or an in vitro biochemical assay that is radioisotope based or nonradioisotope based takes into consideration the type of target, available reagents, assay readout, and availability of automation equipment. With the advent of homogeneous fluorescence based...

Applications Of Human Hepatocytes In Toxicology

Hepatocytes as a Critical Cell Population for Hepatotoxicity Studies A large number of naturally occurring and man-made chemicals are hepatotoxins. In many cases, the toxicity is a function of the metabolic conversion (bioactiva-tion) of the parent compound into highly reactive metabolites. AAP, carbon tetrachloride, dimethylnitrosamine, and halothane are examples of xenobiotics that are ''bioactivated'' by CYP mixed-function monooxygenases in the liver. Species differences in xenobiotic...

Elements of Typical Assays

A basic robotic system for drug screening will usually have the following components robot, servo track, digital gripper, plate storage unit,bar code reader, plate turning station, plate shaker, and liquid dispenser. Depending on the assays to be performed, additional devices will be required. Cell-based assays will require a 37 C temperature controlled CO2 incubator with humidity, and, if cells are being plated by the robotic system, a gentle dispenser, usually with a stirred reservoir to keep...

A A Rationale for New Methods in Drug Discovery

The dramatic increase simultaneously, in the number of targets and the potential hit compounds is the reason for what has been called a change in paradigm in drug discovery 1 . To capitalize fully on the promise of both genomics and combinatorial chemistry, the drug discovery process must change in a fundamental way. Rather than screening a limited number of chemical compounds against a particular target, combinatorial approaches often require screening thousands of compounds. Because of the...

Application Of Recombinant Technology To Cellbased

Integrated Expression of Cloned Genes The accelerated pace of drug screening has led to an increased need for rapid generation of stable cell lines expressing suitable concentrations of the cloned, recombinant targets for the screening efforts. Classical methods for generating stable cell lines have generally relied on the integration of the recombinant gene into random locations within the genome of the host cell. The integrating gene is subjected to unpredictable...

Liquid Transfer

Liquid transfer is a key element of any drug screening method, and virtually every robotic system will have some provisions for the transfer of reagents and other liquids. There are three main types of liquid transfer modes reagent dispensing from bulk reservoirs, single or multichannel transfer between micro plates and small reservoirs, and 96- or 384-multiwell transfer between plates. In each of these categories, syringe pumps are used to perform the liquid transfer because they are more...

Robot Controllers

The early robotic systems for drug screening applications were controlled by the robot controller, usually with the help of another controller for the ''on-off'' devices in the robotic system, such as a PLC (programmable logic controller). Quickly, however, robotic systems became more complex, and it became important to exchange data, such as the weight of a sample, the bar code identification, the volume of liquid to dispense, the reading from an analytical instrument such as a plate reader,...

Highthroughput Screening

The term high-throughput screening (HTS) is used within the drug discovery field to denote the automated robotic systems that test thousands of compounds per week in a targeted assay 5 . Compound screening strategies can be somewhat random, that is, compounds are taken from a library without regard to the compound structure and tested against the selected target(s). Natural products and compounds from older libraries are frequently tested against a wide range of targets in the hopes of...

Multidimensional Readout Technology

Miniaturization requires changes in detection strategies, away from radioactive methods and towards methods based on fluorescence. Presently, radioactivity is used in about half the assays employed in HTS. Many if not most of these assay types have an analogous fluorescence approach, although it is inevitable that particular assays will prove to be difficult or impossible to adapt. Based on currently available data, it is expected that fluorescence methods will be feasible in > 90 of HTS...

Transcriptionbased Screening With Reporter Genes

Alteration in gene expression levels is an indicator of cellular activation. Concentrations of a particular mRNA in a cell can be measured, but such methods are not suitable for HTS. The methodology for measuring changes in gene expression for HTS was facilitated by the advent of both synthetically constructed and naturally derived inducible promoters and response elements. These cis-acting DNA regions respond to intracellular signals, such as changes in levels of cAMP, by increasing...

Cost Justification

The cost justification for automation systems is usually a calculation of the monetary payback over time. The total expenditure for the robotic system is balanced against the cost savings that can be quantified, which usually includes labor, materials, etc. To calculate the cost of the present method of performing the task, the actual time for all of the manual steps is measured and a cost is calculated based upon the cost of the person or persons who are involved with the task. In the case of...

Quality Evaluation

Among other possible advantages of assay miniaturization, one less well addressed issue is the gain of assay quality in drug screening from a statistical point of view. Assay miniaturization makes it possible for each data point to be measured multiple times without consumption of significantly more key reagents. The active compounds (called hits) from a high-throughput random screening process are those statistical ''outliers'' of the screened compounds. The quality of a HTS assay will...

Marcsl

Tion, automation and multiple detection modes, this process can be moved forward in the drug screening process. New readout technologies will enable several parameters to be measured simultaneously, meaning that assays, rather than compounds, will be pooled in a single sample. Just as conventional strategies lead to the pooling of compounds to increase throughput, new detection technologies enable far more information to be obtained in a single measurement, leading to an increase in information...

Robotic Systems

While there is not yet a common high-level programming language for robotic systems, and each supplier has developed its own language, suppliers have improved the ease-of-use of their programming languages, making it easy for operators to learn and use. In general, the languages have moved away from requiring the user to adhere to the programming, command, and syntax rules typical of programming languages. Instead, programming languages have become much more intuitive, allowing the user quickly...

Future Trends

Presently, HTS operations are based on performing the assays within a microtiter plate footprint. The density of the wells within the footprint may vary but the basic mode of operation remains consistent. Simply put, compounds and reagents are mixed within the wells of a plate. Once the reaction is complete some form of detection (e.g., fluorescence, absorption, photon counting) is used to determine the compound's interaction with the reagents. This mode of operation has evolved because of two...

Automated Assays

In order to capitalize on the capabilities of the robotic system, it is highly desirable to run assays that require no human intervention. The manufacturer of the robotic system will work with the user to develop assays that can be performed by mechanical devices that, unfortunately, do not have the benefit of human dexterity or eye-hand coordination. Manufacturers of highly automated laboratory systems will recommend assay procedures that do not compromise the assay yet can be reliably...

Designing Inhibitors Against New Targets

Designing drugs against new targets is a time consuming and expensive process and serious consideration must be given to determining the validity of the protein as a useful drug target 49 . Based upon homology and distinctive motifs belonging to classes of proteins that have traditionally been used as drug targets, thousands of GPCRs, proteases, and kinases have been identified 50 . Many of these newly identified genes code for proteins whose function is unknown and which are known as orphan...

Antifungal And Antibacterial Screens

Some of the earliest examples of molecular genetic screen design come from infectious disease research. Most of the current anti-infective agents in clinical use are fermentation products. In contrast, most other therapeutics (with the possible exception of antitumor agents) are based upon chemically synthesized compounds or mammalian hormones. Rediscovery of known antibiotics is a major issue in natural products screening because of the substantial effort required to purify and characterize...

Others

Zeiss developed an integrated robotic system for high-throughput screening (HTS) and UHTS. This new approach, developed with the pharmaceutical indus try 4 , incorporates a new type of microplate reader, entirely based on microscope technology. In its final release, this detector has 96 parallel microscope optics with a 96-well microplate compatible spacing, enabling the detection of 96-, 384-, and 1536-well plates. Due to the close proximity of the optics to the source of the signal, the...

Software Operating System

Because there are multiple tasks to be performed simultaneously, most robotic systems for drug screening operate with software that operates in a multitasking environment. The controller issues device commands, receives data, and controls the system simultaneously. With today's increasingly complex systems, multitasking capability is a requirement. Due to their cost, availability, reliability, and user familiarity, standard personal computers are frequently used as system controllers. For a...

A EVOscreen

The EVOscreen is a unique platform integrating highly sensitive detection technology and high-precision liquid handling systems in a modular system capable of 100,000 assays per day. A schematic layout of the EVOscreen system is shown in Figure 8a, and a view of the system in operation is shown in Figure 8b. The core elements of the modular system are (1) a high-performance confocal fluorescence detection unit usable in either single-channel or multichannel mode. Proprietary signal processing...

Fluorescent Nanocrystals

There has been a recent interest in the use of biocompatible nanocrystals as in situ fluorescent labels. These microcrystalline particles have interesting fluorescent properties that vary with their composition (CdSe, InP, InAs) and size 70,71 . Their emission spectra are narrow (25-35 nm at half-height) and symmetrical with peak maxima shifting from blue to infrared (460-1800 nm) as the sizes of the particles increase from 2 to 6 nm in diameter. Their emission is long lived (> 100 ns), which...

Confocal Technology

The use of fluorescent dyes has effectively replaced radioactivity as the tagging method of choice for biological assays. The ability to make full use of the fluo rescent molecule and all its properties should be the benchmark of any fluorescence-based assay technology. While most assay strategies make use of only a single fluorescent property, the broad applicability of a screening system requires that flexibility of assay design be extended to the readout technology by including a variety of...

The Microplate

The microtiter plate, now conveniently called ''microplate'' or ''plate,'' deserves a special mention with respect to laboratory automation. In many respects it has been a major determinant of the direction of many products and solutions. And although chip-based technologies are rapidly gaining ground, the microplate will remain to play a prominent role in biopharmaceutical research. The microplate itself is lab automation. It introduced a concept of multiple reaction vessels that could be or...

Screening Of Natural Products

The potential of combinatorial biology was evaluated in a pharmaceutical discovery environment 27,28 with 34 actinomycete strains. The natural products produced by each of these donor strains under defined culture conditions were well characterized with respect to their chemical properties and biological activities. DNA from each donor species was isolated, pooled, cloned, and transferred into the expression host S. lividans. Both natural (vide supra) and combinatorial (vide supra) pathway...

Pros And Cons Of Enzyme Screens

Enzyme-based screens offer advantages due to the measurement of a clearly defined enzyme activity. The development of SAR is more tractable as off-target activities are not confounding (e.g., in whole cell screens the inhibitor must traverse membranes, so permeability and active transport confound potency SAR). Often there is considerable knowledge of active sites or the relevant contact surfaces. In vitro systems allow independent manipulation of conditions to tune the ''sensitivity'' of an...

P

Arm experiences as the arm moves through the work envelop. With either AC or DC servo motors, an encoder sends signals or pulses as the motor moves. When the robot is being taught the movements and positions, the controller counts and stores the motor pulses. In the replay, or automatic mode, the controller tells the motors how many pulses to move, which translates to the speed and stopping position for each axis motor. By duplicating the taught program, the robot performs the tasks exactly as...

Microplate Instrumentation

It is useful to differentiate between stand-alone products, workstations, and robotic systems. In general, a workstation is a device that performs multiple different functions (in our case mostly on microplates). As such, plate washers, dispensers, and readers are considered stand-alone devices, while liquid handlers that combine multiple tools, or that can perform multiple actions on a microplate, should be considered workstations. Robotic systems are a collection of standalone devices and...

Complementation of Homologous Targets

S. cerevisiae-Based Screens for Immunosuppressants Immunosuppressants, such as cyclosporin and FK506, inhibit T cell activation and have made tissue and organ transplantation a reality. These drugs were first identified because of their antifungal activity, and their mechanism of immuno-suppression long remained a mystery. The mechanism of action of cyclosporin and FK506 was determined using S. cerevisiae 20 . Cyclosporin and FK506 bind proteins with peptidyl prolyl isomerase activity (called...

Workstations

Earlier we defined a workstation as a device that performs multiple different functions. The first workstation was the Benchmate from Zymark. This versatile workstation has a dedicated robot arm with a gripper that can move containers to and from different locations on its workbench. Several devices are integrated, such as a precision balance, a single-channel canule for sample transfer, reagent addition, and dilutions, a vortexer, and a HPLC injector. It uses the cylindrical work envelope that...

Cytoplasmic Receptors

Cytoplasmic receptors are ligand binding transcription factors. All except the aryl-hydrocarbon receptor (AhR, a basic helix-loop-helix-PAS bHLH-PAS family member) are members of the classical steroid family, which is now part of the nuclear receptor (NR) superfamily (for recent reviews see Refs. 26-29). Because AhR was originally characterized in biochemical studies as a steroid receptor, other putative steroid receptors may actually be bHLH-PAS structural family members. The biochemical...

Isolation And Culturing Of Human Hepatocytes

The liver is estimated to be approximately 2.6 of the human body weight, consisting of both parenchymal and nonparenchymal cells. The parenchymal cells, commonly known as hepatocytes, comprise the majority of cells of the liver (ap-prox. 60 by cell number and 80 by weight). These cells are the major sites of xenobiotic metabolism and often are the cells injured by chemical toxicants. The nonparenchymal cells include the endothelial cells, which line the sinusoidal space kupffer cells, which are...

Blurring the Lines Between Primary Secondary and Tertiary Screening

Drug screening has traditionally been divided into three phases primary, secondary, and tertiary. As a result of the dramatic increase in targets and compounds to be screened, traditional screening is currently undergoing a re-evaluation. In addition to the move to miniaturization and increases in throughput, increased efficiency is also called for. One way to increase efficiency is to obtain more information at an earlier stage of drug screening. Primary screening is largely the establishment...

B FCS Detection Technologies

Fluorescence correlation spectroscopy (FCS) takes advantage of differences in the translational diffusion of large versus small molecules 7,8 . Each molecule that diffuses through the illuminated confocal focus gives rise to bursts of fluorescent light quanta during the entire course of its journey, and each individual burst is registered. The length of each photon burst corresponds to the time the molecule spends in the confocal focus. The photons emitted in each burst are recorded in a...

Fluorescence Resonance Energy Transfer FRET

One way to increase sensitivity, or perhaps more appropriately, selectivity, is to utilize fluorescence resonance energy transfer (FRET) technology. This technique has seen increasing use in cell imaging, flow cytometry, and confocal microscopy. FRET is the physical process by which energy is transferred from an excited chromophore (donor) to another chromophore (acceptor) by means of intermolec- Figure 4 Titration curves of a compound found to inhibit MMP3 were prepared in a 96-well plate (100...

Hts Using Alginate Macrodroplets

Sodium alginate is the water-soluble salt of alginic acid, a copolymer of 1-4 linked a-D-mannuronic and P-L-guluronic acids, which are extracted from various seaweeds. Sodium alginate solutions have the desirable property of gelling upon contact with divalent cations, such as Ca2+ 26,29 . Ca-alginate has been used as a convenient means of encapsulating cells and enzymes. The physical properties, such as gel strength and porosity, can be controlled by appropriate formulation of the starting...

Miniaturization Of Cellbased Assays Luminescence And Fluorescence

In vitro mammalian cell-based assays constitute an important part of any biomo-lecular screening effort. The main advantage in using whole-cell-based assays is that a cell model may be predictive of the desired physiological response. Data obtained from a cell-based assay not only relates to the target of interest but can on occasion predict such parameters as compound membrane permeability, general cell toxicity, and cell type specificity and selectivity. The disadvantage of using a cell-based...

Modern Integrated Robotic Systems

These systems remain largely based upon the architecture that was originally developed by Zymark. Most progress has been made in the development of appropriate modules or devices and user-friendly graphical (mostly Windows-based) interfaces to system management and scheduling software. While in the original Zymate architecture a proprietary bus interface (the controller) was used exclusively for module interfacing, most systems today are based upon standard industry buses (ranging from RS-232...

Alpha Screen

Gpcr Functional Assay

The amplified luminescent proximity homogeneous assay screen Alpha Screen developed by BioSignal, a Packard Bioscience Company, is a nonradioactive proximity assay using two proprietary 200 nm diameter latex donor and acceptor beads 64 . The beads are coated with hydrogel to prevent nonspecific interactions and self-aggregation and to provide a functionalized surface for cova-lent attachment and to retain dyes. The donor beads contain a photosensitizer that absorbs light at 680 nm laser...

Fluorescence Polarization

Although the theoretical principles of fluorescence polarization FP were described at the beginning of the century 43 , the application of FP to drug discovery, and especially to HTS, became popular only recently 10,44-46 . FP measurements permit analysis of molecular orientation and motion of intrinsically fluorescent or fluorophore-labeled molecules in solution. Therefore FP methodol ogy can be applied to different assays measuring direct binding, such as antibody-antigen interaction,...

Concluding Remarks

Screening of new chemical entities for human pharmacokinetic and toxicological properties using human hepatocytes should greatly enhance the drug develop ment process. Cryopreserved human hepatocytes retain adequate viability and drug-metabolizing enzyme activities 18 , and human hepatocyte-based HrTS assays have been developed. A HrTS assay by definition should have a high capacity and a rapid turnaround time and therefore is not possible with freshly isolated human hepatocytes, as they have...

Aa Aa

Drug Discovery Diagram

Figure 8 Schematic diagram of the HTS in vitro transcription assay. Activator, Tat General factors, general transcription accessory proteins Antibody, anti-RNA DNA PK, proteinase K Oligos, DNA o-nts complementary to target RNA. Figure 8 Schematic diagram of the HTS in vitro transcription assay. Activator, Tat General factors, general transcription accessory proteins Antibody, anti-RNA DNA PK, proteinase K Oligos, DNA o-nts complementary to target RNA. Figure 9 The figure shows data for three...

Functional Expression Of Heterologous Potassium Channels In Yeast

The use of Saccharomyces cerevisiae cells as host for heterologous expression of potassium channels was made possible by advances in the understanding of the regulation of potassium ion homeostasis 5,6 . High-affinity potassium uptake in yeast is mediated by a potassium uptake transporter encoded by TRK1 7 . TRK2 encodes a cation transporter with moderate potassium affinity that contributes significantly to potassium uptake 8,9 . Potassium uptake is determined in part by the electrogenic...

CytostarT Technology

14c Labeled Compounds

Cytostar-T scintillating microplates from Amersham International plc are standard 96-well format, sterile, tissue culture treated microplates. The Cytostar-T plate is a polystyrene plate with a transparent base coated with scintillant. Upon addition of radioactive tracer to cells grown in the bases of the wells, the scintillant generates light when the tracer is bound to the cell membranes or taken up by the cell due to the close proximity of the radioactive isotope to the scintillant at the...