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Results for “High Throughput Screening”

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At least 973 records · Page 54Linked to original sources
PubMed2003

Antibodies in proteomics II: screening, high-throughput characterization and downstream applications.

There are many ways in which the use of antibodies and antibody selection can be improved and developed for high-throughput characterization. Standard protocols, such as immunoprecipitation, western blotting and immunofluorescence, can be used with antibody fragments generated by display technologies. Together with novel approaches, such as antibody chips and intracellular immunization, these methods will yield useful proteomic data following adaptation of the protocols for increased reliability and robustness. To date, most work has focused on the use of standard, well-characterized commercial antibodies. Such protocols need to be adapted for broader use, for example, with antibody fragments or other binders generated by display technologies, because it is unlikely that traditional approaches will provide the required throughput.

Antibodies↗
PubMed2000

Combining high-throughput pharmacokinetic screens at the hits-to-leads stage of drug discovery.

Profound technological advances in the drug discovery process have led to the identification of an increasingly large number of promising compounds at the hits-to-leads stage. Higher-throughput pharmacokinetic screens have therefore been developed to enhance the tractability of selected leads and minimize the risk of failure in the later stages of drug development.

Journal Article↗
PubMedDate not supplied

Innovative modular membrane adsorber system for high-throughput downstream screening for protein purification.

To develop the most efficient strategy for the purification of proteins, two types of adsorber membrane devices with different functionalities were designed and tested: 8-strips and single spin columns. The most suitable type of membrane adsorber and the optimal chromatographic loading/elution conditions for several target proteins from different biological matrices could be determined simultaneously in microliter scale. Ion exchange (IEX), metal chelate (MC), and Concanavalin A (Con A) modified membrane types were tested in the devices. Bovine serum albumin (BSA) and lysozyme were used as model proteins for investigations of the binding capacity and protein recovery percentage of the 8-strip anion exchange and the cation exchange membrane. The isolation of His(6)-tagged proteins, Bgl-His and GFP-His from fermentation broth and lysate, respectively, was performed using an 8-strip metal chelate affinity membrane loaded with different metal ions. Separation behavior of a ternary protein mixture (BSA, lysozyme, and Bgl-His) was studied in 8-strips IEX and metal chelate membrane chromatography. The Con A affinity devices were developed on the basis of metal chelate membrane spin columns loaded with Cu(2+) ions and investigated using glucose oxidase (GOD) as model protein. In summary, the advantages of the membrane adsorber technology, such as fast processing and easy scale-up, were utilized. The devices made it possible to load the membrane directly with preclarified fermentation broth or cell lysate and separate the protein of interest often in a single step.

Adsorption↗
PubMed2004

Efficient method for high-throughput virtual screening based on flexible docking: discovery of novel acetylcholinesterase inhibitors.

A method of easily finding ligands, with a variety of core structures, for a given target macromolecule would greatly contribute to the rapid identification of novel lead compounds for drug development. We have developed an efficient method for discovering ligand candidates from a number of flexible compounds included in databases, when the three-dimensional (3D) structure of the drug target is available. The method, named ADAM&EVE, makes use of our automated docking method ADAM, which has already been reported. Like ADAM, ADAM&EVE takes account of the flexibility of each molecule in databases, by exploring the conformational space fully and continuously. Database screening has been made much faster than with ADAM through the tuning of parameters, so that computational screening of several hundred thousand compounds is possible in a practical time. Promising ligand candidates can be selected according to various criteria based on the docking results and characteristics of compounds. Furthermore, we have developed a new tool, EVE-MAKE, for automatically preparing the additional compound data necessary for flexible docking calculation, prior to 3D database screening. Among several successful cases of lead discovery by ADAM&EVE, the finding of novel acetylcholinesterase (AChE) inhibitors is presented here. We performed a virtual screening of about 160 000 commercially available compounds against the X-ray crystallographic structure of AChE. Among 114 compounds that could be purchased and assayed, 35 molecules with various core structures showed inhibitory activities with IC(50) values less than 100 microM. Thirteen compounds had IC(50) values between 0.5 and 10 microM, and almost all their core structures are very different from those of known inhibitors. The results demonstrate the effectiveness and validity of the ADAM&EVE approach and provide a starting point for development of novel drugs to treat Alzheimer's disease.

Acetylcholinesterase↗
PubMed2006

High-throughput RNAi screening in cultured cells: a user's guide.

RNA interference has re-energized the field of functional genomics by enabling genome-scale loss-of-function screens in cultured cells. Looking back on the lessons that have been learned from the first wave of technology developments and applications in this exciting field, we provide both a user's guide for newcomers to the field and a detailed examination of some more complex issues, particularly concerning optimization and quality control, for more advanced users. From a discussion of cell lines, screening paradigms, reagent types and read-out methodologies, we explore in particular the complexities of designing optimal controls and normalization strategies for these challenging but extremely powerful studies.

Animals↗
PubMed2000

Functional importance of the Ala(116)-Pro(136) region in the calcium-sensing receptor. Constitutive activity and inverse agonism in a family C G-protein-coupled receptor.

The calcium-sensing receptor (CaR) belongs to family C of the G-protein-coupled receptor superfamily. To date 14 activating mutations in CaR showing increased sensitivity to Ca(2+) have been identified in humans with autosomal dominant hypocalcemia. Four of these activating mutations are found in the Ala(116)-Pro(136) region of CaR, indicating that this part of the receptor is particularly sensitive to mutation-induced activation. This region was subjected to random saturation mutagenesis, and 219 mutant receptor clones were isolated and screened pharmacologically in a high throughput screening assay. Selected mutants were characterized further in an inositol phosphate assay. The vast majority of the mutants tested displayed an increased affinity for Ca(2+). Furthermore, 21 of the mutants showed increased basal activity in the absence of agonist. This constitutive activity was not diminished when the mutations were transferred to a chimeric receptor Ca/1a consisting of the amino-terminal domain of the CaR and the 7 transmembrane and intracellular domains of the metabotropic glutamate receptor mGluR1a. CPCCOEt, a noncompetitive antagonist acting at the 7 transmembrane domain of mGluR1a, suppressed the elevated basal response of the constitutively activated Ca/1a mutants demonstrating inverse agonist activity of CPCCOEt. Taken together, our results demonstrate that the Ala(116)-Pro(136) region is of key importance for the maintenance of the inactive conformation of CaR.

3T3 Cells↗
PubMed2006

Novel yeast cell-based assay to screen for inhibitors of human cytomegalovirus protease in a high-throughput format.

The protease encoded by the human cytomegalovirus (HCMV) is an attractive target for antiviral drug development because of its essential function in viral replication. We describe here a cellular assay in the yeast Saccharomyces cerevisiae for the identification of small molecule inhibitors of HCMV protease by conditional growth in selective medium. In this system, the protease cleavage sequence is inserted into the N-(5'-phosphoribosyl)anthranilate isomerase (Trp1p), a yeast protein essential for cell proliferation in the absence of tryptophan. Coexpression of HCMV protease with the engineered Trp1p substrate in yeast cells results in site-specific cleavage and functional inactivation of the Trp1p enzyme, thereby leading to an arrest of cell proliferation. This growth arrest can be suppressed by the addition of validated HCMV protease inhibitors. The growth selection system presented here provides the basis for a high-throughput screen to identify HCMV protease inhibitors that are active in eukaryotic cells.

Aldose-Ketose Isomerases↗
PubMed2005

Psi-screen, an in vitro toxicity test system: applications in the bioassay of perfumes and fragrance chemicals.

The effects of 65 perfume formulations (perfume oils, perfumes, eau de parfum, eau de toilette) on mitochondrial membrane potential (Psim) and mitochondrial respiration have been investigated using a mitochondria-based assay for (Psim, termed Psi-Screen. All the perfume formulations tested are highly active in the Psi-Screen assay, and the major site of inhibition in all cases is NADH-ubiquinone reductase (Complex I). This is confirmed in studies on the inhibition of NADH oxidase and NADH-ubiquinone reductase. Some formulations also inhibit succinate oxidation at either Complex II or Complex III. Evidence for the inhibition of mitochondrial ATPase is presented, as well as for the induction of reactive oxygen species production by perfume inhibition of Complex I. Thus, perfume formulations are multiple inhibitor mixtures which inhibit multiple bioenergetic functions at high dilutions. The implications of these findings are discussed with respect to cell toxicity via necrosis and/or apoptosis. Twenty candidate fragrance chemicals were investigated and all inhibited Complex I (5 at <35 microM). Mass screening strategies and high-throughput screening assays are discussed.

Animals↗
PubMed2000

Miniaturization of a functional transcription assay in yeast (human progesterone receptor) in the 384- and 1536-well plate format.

Miniaturization of high throughput screening assays to high-density microplate formats (384 or 1536 wells) is currently the focus of research activity in modern drug discovery facilities. In this article, we describe the adaptation of a fluorescence-based functional transcription assay in yeast for assessing modulators of human progesterone receptor to the 384- and 1536-well microplate format, comparing the experimental results to those obtained in the well-established 96-well format. The experiences gained from the optimization of the liquid-handling procedures and the miniaturization of an enzyme assay (beta-galactosidase) were implemented. Thus optimized pipetting protocols were developed to perform a reporter gene assay in yeast in microplate formats of higher density. In the functional transcription assay in yeast, the reporter gene expression showed the expected dependence on the ligand's dose and affinity in principle in all three microplate formats. For the first time, this assay system has been established in the 1536-well microplate format using CyBi-Well 96/384/1536 as the liquid-handling unit. The comparison of the signal:background ratios showed a lower sensitivity of the assay in the microplate formats of higher density. This study is an example of a successful miniaturization of a yeast cell-based assay to high-density plate formats on the basis of a careful adaptation procedure and optimized liquid-handling conditions.

Cloning, Molecular↗
PubMed2004

A generally applicable, high-throughput screening-compatible assay to identify, evaluate, and optimize antimicrobial agents for drug therapy.

Efficacy and tolerability are the key criteria for a successful medication in the clinic. Therefore, a new test method to obtain selective and active lead molecules has been developed. Recently, this novel screening strategy enabled a breakthrough in drug discovery in the field of herpes viruses. Here the authors report that this assay is a generally applicable screening test, which allows not only for identifying tolerable and potent antimicrobial agents in compound libraries, but also covers all potential in vitro targets of both the pathogen and the host simultaneously. The test system mimics the smallest unit of a natural infection. Host cells are incubated in the presence of the test sample and are infected with microbes, such as viruses, bacteria, or fungi. Analogous to (lethal challenge) animal models, cell survival is determined. This assay maximizes the chances of success of anti-infective drug discovery, is sensitive, robust, time- and cost-efficient, and especially effective in optimizing screening hits to lead structures and development candidates. In addition to the minimal inhibitory concentration or dose, this test system simultaneously provides the selectivity index, a measure of tolerability in vitro. The authors propose the activity selectivity assay format as a new standard in anti-infective drug discovery and clinical development.

Animals↗
PubMed2006

High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources.

To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.

Cell Count↗
PubMed2006

Protein chips for high-throughput doping screening in athletes.

Sport and doping are a contradiction in terms, however, doping abuse in sports has been a serious problem for many years. The systematic screening of every athlete for all prohibited drugs should be an indispensable feature of the Olympic Games. The gas chromatography mass spectrometry method is reserved as a reference method, but is limited by its low throughput. The advent of protein chip technology may enable the screening of all athletes for any illegal use of drugs.

Doping in Sports↗
PubMed2005

Improved T-DNA vector for tagging plant promoters via high-throughput luciferase screening.

Transferred DNA (T-DNA) tagging is a powerful tool for tagging and in planta characterization of plant genes on a genome-wide scale. An improved promoter tagging vector is described here, which contains the codon-optimized luciferase (luc+) reporter gene 31 bp from the right border of the T-DNA. Compared to the wild-type luciferase gene, this construct provides significantly increased reporter gene expression and a 40 times higher tagging frequency. The utility of the construct is demonstrated in banana, a tropical monocot species, by screening embryogenic cell colonies and regenerated plants with an ultrasensitive charged-coupled device (CCD) camera. The improved vector resulted in a luciferase activation frequency of 2.5% in 19,000 cell colonies screened. Detailed molecular analysis of flanking DNA sequences in a tagged line revealed insertion of the luciferase tag in a novel gene with near-constitutive expression.

Base Sequence↗
PubMed2000

High throughput genetic screening for the detection of hereditary non-polyposis colon cancer (HNPCC) using capillary electrophoresis.

Approximately 5-10% of all colorectal carcinomas arise from cancer predisposition syndromes caused by heterozygote germline mutations in post-replicative DNA mismatch repair (MMR) genes. In contrast to gastrointestinal polyposis syndromes, carcinomas in these patients do not occur on the background of increased numbers of polyps and hence are refered to as hereditary non-polyposis colorectal cancers (HNPCC). Six different MMR genes, MSH2, MSH3, MSH6, MLH1, MLH3 and PMS2, have been identified in the human genome. In the majority of HNPCC patients, heterozygote germline mutations are present in the MSH2 or MLH1 gene. Detection of mutations by conventional sequencing technology is expensive and labor intensive due to the complex intron and/or exon structures. In this study, we therefore have explored whether capillary electrophoresis-based single strand conformation polymorphism (SSCP-CE) provides a reliable means for mutation screening. We have tested different MLH1 mutations in exons 9 and 16 and find that SSCP-CE produces reliable electrophoretic patterns that allow recognition of wild-type alleles, microdeletions and point mutations. In summary, SSCP-CE provides a rapid, automated, and cost-effective technology for MSH2 and MLH1 mutation screening and will facilitate genetic diagnostics for HNPCC patients.

Colonic Neoplasms↗