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PubMed1997

High-throughput screening: advances in assay technologies.

Both isotopic and nonisotopic assay methodologies are employed in high-throughput screening for drug discovery. Recent advances in cell-based and in vitro biochemical assays will be reviewed, with special emphasis on detection technologies amenable to automated 'mix and read' procedures in high-throughput screening. A major trend is the advent of homogenous assay systems which employ fluorescence resonance energy transfer, fluorescence polarization, and fluorescence correlation spectroscopy. Cell-based assay systems have also become popular in high-throughput screens in which active compounds that directly modulate the disease target are identified. Colorimetric and amperometric methods have also been described recently, but are yet to be adapted widely in high-throughput screens.

Colorimetry↗

PubMed2005

Validation of a high-throughput absorption, distribution, metabolism, and excretion (ADME) system and results for 60 literature compounds.

A high-throughput analytical system for the support of absorption, distribution, metabolism, and excretion (ADME) was constructed in collaboration with Gubbs Inc. to commercialize corresponding software. We sought to quickly build and validate this system for microsomal clearance assessment using 60 commercially available non-proprietary compounds that are non-DEA-restricted in addition to 36 proprietary Millennium compounds that had already been assessed using a low-throughput infrastructure. The system was constructed such that a approximately 45 second total cycle time was achieved injection-to-injection. Software was successfully coded to enable the analyst to submit multiple batches, and modify multiple methods very quickly for use with Applied Biosystems Analyst 1.3. After acquisition the software was used to simultaneously integrate multiple injection chromatograms, regress the data, and calculate clearance such that all of the data could be easily and immediately reviewed by both bioanalytical and enzymology personnel. Unfortunately, despite an exhaustive search of the literature, we were unable to find a large number of non-proprietary compound data for validation, so we provide such a source of data here. Results are presented for the 60 literature compounds that were assessed. A good correlation was observed between literature results for 16 compounds that we were able to find and the results obtained using the system. The Millennium proprietary compounds that we assessed using both low- and high-throughput approaches also correlated well. We present here a system for the support of high-throughput in vitro ADME analysis and also present the results of 60 non-proprietary, non-DEA-scheduled compounds to facilitate the validation efforts of others. Finally, we present commercially available software to facilitate high-throughput ADME systems in the community.

Absorption↗

PubMed1997

High-throughput microsatellite analysis using fluorescent dUTPs for high-resolution genetic mapping of the mouse genome.

The use of fluorescent end-labeled primers has proved successful for rapid, semiautomated genotyping of microsatellite loci. However, custom synthesis is expensive and costs can be prohibitive when a wide range of markers is to be analyzed for only a few genotypings. This particularly applies to high-resolution genetic mapping in the mouse either in the construction of global maps or in the production of local high-resolution genetic maps for positional cloning. We demonstrate here the use of fluorescent dUTPs for cost-effective, high-throughput microsatellite genotyping in the mouse. This alternative to the use of fluorescent end-labeled primers for semiautomated genotyping is potentially applicable to the construction of linkage maps in other species.

Animals↗

PubMed2004

High-throughput multiplexed capillary electrophoresis in drug discovery.

The demand for high-throughput analytical tools to support drug discovery applications has led to the development of multiplexed capillary electrophoresis and multichannel microfluidic devices to characterize libraries of compounds and alleviate backlogs in the discovery process. The capability to analyze multiple samples in parallel, and the diverse separation conditions that are permissible, facilitates rapid turnaround times. Examples of high-throughput applications of multiplexed electrophoresis in drug discovery include: physicochemical profiling, enzyme analysis, chiral separations and protein/metabolite analysis. Many single capillary electrophoresis methods can be potentially adapted to a multiplexed format, therefore, we anticipate the development of other high-throughput applications in the near future, which should facilitate decreases in sample analysis time and help improve laboratory efficiency.

Drug Design↗

PubMed2004

Ice-cap. A high-throughput method for capturing plant tissue samples for genotype analysis.

High-throughput genotype screening is rapidly becoming a standard research tool in the post-genomic era. A major bottleneck currently exists, however, that limits the utility of this approach in the plant sciences. The rate-limiting step in current high-throughput pipelines is that tissue samples from living plants must be collected manually, one plant at a time. In this article I describe a novel method for harvesting tissue samples from living seedlings that eliminates this bottleneck. The method has been named Ice-Cap to reflect the fact that ice is used to capture the tissue samples. The planting of seeds, growth of seedlings, and harvesting of tissue are all performed in a 96-well format. I demonstrate the utility of this system by using tissue harvested by Ice-Cap to genotype a population of Arabidopsis seedlings that is segregating a previously characterized mutation. Because the harvesting of tissue is performed in a nondestructive manner, plants with the desired genotype can be transferred to soil and grown to maturity. I also show that Ice-Cap can be used to analyze genomic DNA from rice (Oryza sativa) seedlings. It is expected that this method will be applicable to high-throughput screening with many different plant species, making it a useful technology for performing marker assisted selection.

Genetic Techniques↗

PubMed2003

A homogeneous high-throughput genotyping method based on competitive hybridization.

OBJECTIVES: A reliable high-throughput assay system is necessary for the analysis of the ever-increasing numbers of single-nucleotide polymorphisms (SNP) relevant to genetic screening studies. We describe an assay suitable also for large-scale screening programs. DESIGN AND METHODS: The one-step assay is based on asymmetric PCR amplification of the target sequence and subsequent time-resolved fluorescence measurement. Asymmetric amplification results in a single-stranded PCR product that is detected in the amplification vessel with a highly sensitive, homogeneous hybridization method. RESULTS: A dual label, homogeneous high-throughput platform for nucleic acid sequence analysis was developed and validated using a C/T single-nucleotide polymorphism in the insulin gene as a model analyte and applied also to two other SNP-assays (poliovirus receptor A/G-polymorphism and CD86-gene exon 2 A/G-polymorphism). CONCLUSIONS: The described high-throughput genotyping technology is very competitive in price, simple in design and easily applied to any analyte sequence.

Genetic Testing↗

PubMed2000

A strategy for primary high throughput cytotoxicity screening in pharmaceutical toxicology.

PURPOSE: Recent advances in combinatorial chemistry and high throughput screens for pharmacologic activity have created an increasing demand for in vitro high throughput screens for toxicological evaluation in the early phases of drug discovery. METHODS: To develop a strategy for such a screen, we have conducted a data mining study of the National Cancer Institute's Developmental Therapeutics Program (DTP) cytotoxicity database. RESULTS: Using hierarchical cluster analysis, we confirmed that the different tissues of origin and individual cell lines showed differential sensitivity to compounds in the DTP Standard Agents database. Surprisingly, however, approaching the data globally, linear regression analysis showed that the differences were relatively minor. Comparison with the literature on acute toxicity in mice showed that the predictive power of growth inhibition was marginally superior to that of cell death. CONCLUSIONS: This datamining study suggests that in designing a strategy for high throughput cytotoxicity screening: a single cell line, the choice of which may not be critical, can be used as a primary screen; a single end point may be an adequate measure and a cut off value for 50% growth inhibition between 10(-6) and 10(-8) M may be a reasonable starting point for accepting a cytotoxic compound for scale up and further study.

Animals↗

PubMed2002

Development of combinatorial chemistry methods for coatings: high-throughput optimization of curing parameters of coatings libraries.

An automated analytical system has been implemented for the high-throughput optimization of processing conditions such as curing parameters in fabrication of UV-cured automotive organic protective coatings. Selection of optimum process conditions of combinatorial arrays of coatings is essential to correlate the high-throughput screening and conventional processes and to achieve the desired physical properties of coatings. For monitoring of curing conditions of each coating in the array, a viscosity-sensitive fluorophore 4,4'-bis(2-benzoxazolyl)stilbene was incorporated into coating formulations. This fluorescence tagging approach permitted us to combine a gradient temperature heater and a UV curing system with the full capabilities of our high-throughput screening system, including generation of spectroscopic data and its analysis. This investigation demonstrated the possibility of rapid decoupling of temperature and radiation effects in curing of UV-curable coating formulations by using multiple coatings and process conditions at once. While the system described here was implemented for high-throughput optimization of temperature conditions of radiation curing of arrays of organic protective coatings for automotive applications, this system can be further applied for a variety of other applications where optimization of process parameters can be studied in situ or off-line using optical spectroscopic tools.

Journal Article↗

PubMed2005

High-throughput magnetic resonance imaging in mice for phenotyping and therapeutic evaluation.

High-throughput mouse magnetic resonance imaging (MRI) is seeing rapidly increasing demand in development of therapeutics. Recent advances including higher-field systems, new gradient and radio frequency coils and new pulse sequences, coupled with efficient animal preparation and data handling, allow high-throughput MRI under certain protocols. However, with current shifts from anatomic to functional and molecular imaging, innovative technology is required to meet new throughput demands. The first multiple mouse imaging strategies have provided a glimpse of the future state-of-the-art. However, the successful translation of standard clinical MRI technology to preclinical MRI is required to facilitate next-generation high-throughput MRI.

Animals↗

PubMed2000

High-throughput screening for human collagenase 1 inhibitors.

AIM: To establish a high-throughput method for inhibitor screening using a recombinant collagenase catalytic domain. METHODS: Human collagenase 1 catalytic domain protein was expressed in E coli and used for screening a set of 2720 compounds in a high-throughput fashion. RESULTS: The screening was accomplished within 2 h and 10 min with consumption of each compound at 4 micrograms. Sixty-six compounds were identified with > 60% inhibitory activity at 20 mg/L, among which 44 compounds were confirmed by subsequent testing at multiple concentrations. The most potent compound showed an IC50 at 4.3 mumol/L, and there were total 15 compounds with IC50 less than 20 mumol/L. CONCLUSION: The high-throughput method using the recombinant collagenase is fast, effective and practical in identifying inhibitors.

Collagenases↗

PubMed2000

Direct plasma sample injection in multiple-component LC-MS-MS assays for high-throughput pharmacokinetic screening.

The simultaneous dosing of numerous compounds followed by multiple-component analysis using LC-MS-MS (the N-in-1 approach) has significantly improved the throughput of the drug-screening process. However, plasma samples still need to be extracted before LC-MS-MS analysis, which frequently limits the throughput of the assay. In this work, a high-throughput on-line extraction technique has been developed for multiple-component LC-MS-MS assays using a high-flow column-switching technique. In N-in-1 LC-MS-MS assays, high sensitivity is required since the dose level is generally reduced to minimize drug-drug interactions. In addition, good chromatographic separation is essential to minimize interference and suppression effects. The direct plasma sample injection method developed in this work has successfully met the two requirements for multiple-component LC-MS-MS assays in high-throughput pharmacokinetic screening. Plasma samples containing a large number of potential drug candidates were directly injected onto an extraction column operated under a flow rate sufficiently high to exhibit a turbulent-flow profile. The extracted analytes were then eluted onto an analytical column via column switching for LC-MS-MS analysis. The use of turbulent flow resulted in a faster and more rugged extraction with reduced carryover compared with results obtained under laminar-flow conditions. Meanwhile, the use of a column-switching method maintained the chromatographic resolving power and high sensitivity of the LC-MS-MS assay. Separation efficiency, dynamic range, accuracy, and precision comparable with those of solid-phase extraction have been achieved with the turbulent-flow column-switching technique. As a result, this technique has been successfully and routinely used for high-throughput pharmacokinetic screening.

Animals↗

PubMed2004

Disposable chromatography for a high-throughput nano-ESI/MS and nano-ESI/MS-MS platform.

High-throughput proteomics has typically relied on protein identification based on MALDI-MS peptide maps of proteolytic digests of 2D-gel-separated proteins. This technique, however, requires significant sequence coverage in order to achieve a high level of confidence in the identification. Tandem MS data have the advantage of requiring fewer peptides (2) for high confidence identification, assuming adequate MS/MS sequence coverage. MALDI-MS/MS techniques are becoming available, but can still be problematic because of the difficulty of inducing fragment ions of a singly charged parent ion. Electrospray ionization, however, has the advantage of generating multiply charged species that are more readily fragmented during MS/MS analysis. Two electrospray/tandem mass spectrometry-based approaches, nanovial-ESI-MS/MS and LC-MS/MS, are used for high throughput proteomics, but much less often than MALDI-MS and peptide mass fingerprinting. Nanovial introduction entails extensive manual manipulation and often shows significant chemical background from the in-gel digest. LC-MS has the advantages that the chemical background can be removed prior to analysis and the analytes are concentrated during the separation, resulting in more abundant analyte signals. On the other hand, LC-MS can often be time intensive. Here, we report the incorporation of on-line sample clean-up and analyte concentration with a high-throughput, chip-based, robotic nano-ESI-MS platform for proteomics studies.

Angiotensinogen↗

PubMed2005

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.

BACKGROUND: High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. RESULTS: Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that are used in the high-throughput community to determine assay robustness (Z'-value) demonstrate the suitability of this format for high-throughput screening applications for detection of inhibitors of enzyme activity. CONCLUSION: The QTL Lightspeed protein detection system provides a simple mix and measure "turn on" assay for the detection of kinase activity using natural protein substrates. The platform is robust and allows for identification of inhibitors of kinase activity.

Adaptor Proteins, Signal Transducing↗

PubMed2001

High-throughput techniques for compound characterization and purification.

A new paradigm in drug discovery is the synthesis of structurally diverse collections of compounds, so-called libraries, followed by high-throughput biological screening. High-throughput characterization and purification techniques are required to provide high-quality compounds and reliable biological data, which has led to the development of faster methods, system automation and parallel approaches. This review summarizes recent advances in support of analytical characterization and preparative purification technologies. Notably, mass spectrometry (MS) and supercritical fluid chromatography (SFC) are among the areas where new developments have had a major impact on defining these high-throughput applications.

Chromatography↗

PubMed1994

The genome reconstruction manager: a software environment for supporting high-throughput DNA sequencing.

A new software system designed for use in high-throughput DNA sequencing laboratories is described. The Genome Reconstruction Manager (GRM) was developed from requirements derived from ongoing large-scale DNA sequencing projects. Object-oriented principles were followed in designing the system, and tools supporting object-oriented system development were employed for its implementation. GRM provides several advances in software support for high-throughput DNA sequencing: support for random, directed, and mixed sequencing strategies; a novel system for fragment assembly; a commercial object data-base management system for data storage; a client/server architecture for using network computational servers; and an underlying data model that can evolve to support fully automatic sequence reconstruction. GRM is currently being deployed for production use in high-throughput DNA sequencing projects.

Algorithms↗

PubMed2000

From genes to proteins: high-throughput expression and purification of the human proteome.

The development of high-throughput methods for gene discovery has paved the way for the design of new strategies for genome-scale protein analysis. Lawrence Livermore National Laboratory and Onyx Pharmaceuticals, Inc., have produced an automatable system for the expression and purification of large numbers of proteins encoded by cDNA clones from the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) collection. This high-throughput protein expression system has been developed for the analysis of the human proteome, the protein equivalent of the human genome, comprising the translated products of all expressed genes. Functional and structural analysis of novel genes identified by EST (Expressed Sequence Tag) sequencing and the Human Genome Project will be greatly advanced by the application of this high-throughput expression system for protein production. A prototype was designed to demonstrate the feasibility of our approach. Using a PCR-based strategy, 72 unique IMAGE cDNA clones have been used to create an array of recombinant baculoviruses in a 96-well microtiter plate format. Forty-two percent of these cDNAs successfully produced soluble, recombinant protein. All of the steps in this process, from PCR to protein production, were performed in 96-well microtiter plates, and are thus amenable to automation. Each recombinant protein was engineered to incorporate an epitope tag at the amino terminal end to allow for immunoaffinity purification. Proteins expressed from this system are currently being analyzed for functional and biochemical properties.

Cloning, Molecular↗

PubMed2004

Predicting gene function through systematic analysis and quality assessment of high-throughput data.

MOTIVATION: Determining gene function is an important challenge arising from the availability of whole genome sequences. Until recently, approaches based on sequence homology were the only high-throughput method for predicting gene function. Use of high-throughput generated experimental data sets for determining gene function has been limited for several reasons. RESULTS: Here a new approach is presented for integration of high-throughput data sets, leading to prediction of function based on relationships supported by multiple types and sources of data. This is achieved with a database containing 125 different high-throughput data sets describing phenotypes, cellular localizations, protein interactions and mRNA expression levels from Saccharomyces cerevisiae, using a bit-vector representation and information content-based ranking. The approach takes characteristic and qualitative differences between the data sets into account, is highly flexible, efficient and scalable. Database queries result in predictions for 543 uncharacterized genes, based on multiple functional relationships each supported by at least three types of experimental data. Some of these are experimentally verified, further demonstrating their reliability. The results also generate insights into the relative merits of different data types and provide a coherent framework for functional genomic datamining. AVAILABILITY: Free availability over the Internet. CONTACT: f.c.p.holstege@med.uu.nl SUPPLEMENTARY INFORMATION: http://www.genomics.med.uu.nl/pub/pk/comb_gen_network.

Computer Simulation↗

PubMed2004

Application of chemoinformatics to high-throughput screening: practical considerations.

The objective of this chapter is to summarize and evaluate some of the most common chemoinformatic methods that are applied to the analysis of high-throughput-screening data. The chapter will briefly describe current high-throughput-screening practices and will stress how the major constraint on the application of chemoinformatics is often the quality of high-throughput-screening data. Discussion of the NCI dataset and how it differs from most high-throughput-screening datasets will be made to highlight this point.

Chemical Phenomena↗