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At least 1,459 records · Page 81Linked to original sources
PubMed2000

Hematin polymerization assay as a high-throughput screen for identification of new antimalarial pharmacophores.

Hematin polymerization is a parasite-specific process that enables the detoxification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a unique pharmacological drug target. We have developed a high-throughput in vitro microassay of hematin polymerization based on the detection of (14)C-labeled hematin incorporated into polymeric hemozoin (malaria pigment). The assay uses 96-well filtration microplates and requires 12 h and a Wallac 1450 MicroBeta liquid scintillation counter. The robustness of the assay allowed the rapid screening and evaluation of more than 100, 000 compounds. Random screening was complemented by the development of a pharmacophore hypothesis using the "Catalyst" program and a large amount of data available on the inhibitory activity of a large library of 4-aminoquinolines. Using these methods, we identified "hit" compounds belonging to several chemical structural classes that had potential antimalarial activity. Follow-up evaluation of the antimalarial activity of these compounds in culture and in the Plasmodium berghei murine model further identified compounds with actual antimalarial activity. Of particular interest was a triarylcarbinol (Ro 06-9075) and a related benzophenone (Ro 22-8014) that showed oral activity in the murine model. These compounds are chemically accessible and could form the basis of a new antimalarial medicinal chemistry program.

Animals↗
PubMed2003

Luminescence resonance energy transfer-based high-throughput screening assay for inhibitors of essential protein-protein interactions in bacterial RNA polymerase.

The binding of sigma factors to core RNA polymerase is essential for the specific initiation of transcription in eubacteria and is thus critical for cell growth. Since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic RNA polymerases, sigma factor binding is a promising target for drug discovery. A homogeneous assay for sigma binding to RNA polymerase (Escherichia coli) based on luminescence resonance energy transfer (LRET) was developed by using a europium-labeled sigma70 and an IC5-labeled fragment of the beta' subunit of RNA polymerase (amino acid residues 100 through 309). Inhibition of sigma binding was measured by the loss of LRET through a decrease in IC5 emission. The technical advances offered by LRET resulted in a very robust assay suitable for high-throughput screening, and LRET was successfully used to screen a crude natural-product library. We illustrate this method as a powerful tool to investigate any essential protein-protein interaction for basic research and drug discovery.

Animals↗
PubMed2006

Creation of novel Protein Transduction Domain (PTD) mutants by a phage display-based high-throughput screening system.

Significant research effort is currently focused on Protein Transduction Domains (PTDs) as potential intracellular drug delivery carriers. However, the application of this technology is limited because the transduction efficiencies are often insufficient for therapeutic purposes, even using HIV-1 Tat peptide. Here we describe a high-throughput screening method based on a phage display system for isolating novel PTDs with improved cell penetration activity. The screening method involves using protein synthesis inhibitory factor (PSIF) as cargo of PTD. Using this method, several Tat-PTD mutants of superior cell-penetrating activity were isolated. Interestingly, the amino acid sequence of the PTD mutants contained some characteristic residues, such as proline. Thus, our screening method may prove useful in determining the relationship between protein transduction and amino acid sequence.

Bacteriophages↗
PubMed2002

Multiplex gene expression analysis for high-throughput drug discovery: screening and analysis of compounds affecting genes overexpressed in cancer cells.

Drug discovery strategies are needed that can rapidly exploit multiple therapeutic targets associated with the complex gene expression changes that characterize a polygenic disease such as cancer. We report a new cell-based high-throughput technology for screening chemical libraries against several potential cancer target genes in parallel. Multiplex gene expression (MGE) analysis provides direct and quantitative measurement of multiple endogenous mRNAs using a multiplexed detection system coupled to reverse transcription-PCR. A multiplex assay for six genes overexpressed in cancer cells was used to screen 9000 chemicals and known drugs in the human prostate cancer cell line PC-3. Active compounds that modulated gene expression levels were identified, and IC50 values were determined for compounds that bind DNA, cell surface receptors, and components of intracellular signaling pathways. A class of steroids related to the cardiac glycosides was identified that potently inhibited the plasma membrane Na(+)K(+)-ATPase resulting in the inhibition of four of the prostate target genes including transcription factors Hoxb-13, hPSE/PDEF, hepatocyte nuclear factor-3alpha, and the inhibitor of apoptosis, survivin. Representative compounds selectively induced apoptosis in PC-3 cells compared with the nonmetastatic cell line BPH-1. The multiplex assay distinguished potencies among structural variants, enabling structure-activity analysis suitable for chemical optimization studies. A second multiplex assay for five toxicological markers, Hsp70, Gadd153, Gadd45, O6-methylguanine-DNA methyltransferase, and cyclophilin, detected compounds that caused DNA damage and cellular stress and was a more sensitive and specific indicator of potential toxicity than measurement of cell viability. MGE analysis facilitates rapid drug screening and compound optimization, the simultaneous measurement of toxicological end points, and gene function analysis.

Actins↗
PubMed2003

Electrospray ionization tandem mass spectrometry in high-throughput screening of homogeneous catalysts.

The frontispiece shows an illustration by John Tenniel and an excerpt from the 1865 edition of Lewis Carroll's "Alice in Wonderland". Because everything in Wonderland runs counter to logic, the Queen of Hearts declares in Alice's trial "Sentence first-verdict afterwards". High-throughput screening of catalysts, as it is conventionally practiced, does "Synthesis first-screening afterwards" which, as is argued in this review, also backwards. Given the particular constraints present in organometallic complexes, it is more efficient to develop a selective synthesis only when it has already been determined that a structure is likely to be better. The consequence is that screening methods must be able to handle ill-defined mixtures. Electrospray ionization tandem mass spectrometry is presented as a technical solution to this problem.

Journal Article↗
PubMed2004

A high-throughput fluorescence polarization assay for signal transducer and activator of transcription 3.

Signal transducer and activator of transcription 3 (STAT3) is overactive in a wide variety of human tumors. Activity of STAT3 requires its own SH2-domain-mediated binding to phosphotyrosine-containing sequences. We have developed a high-throughput binding assay, based on fluorescence polarization, which allows screening for small molecules that bind to the STAT3 SH2 domain and thereby inhibit its activity. The basis of this assay is the binding of a fluorescein-labeled phosphotyrosine-peptide derived from the interleukin-6 receptor subunit gp130 to unphosphorylated STAT3 with a K(d) of 150 nM. The assay is stable with regard to salt concentration, dimethyl sulfoxide concentration, and time. It has been adapted to a 384-well format, with a Z' value of 0.87, and can be used to screen for small molecules that bind to the STAT3 SH2 domain.

Antigens, CD↗
PubMedDate not supplied

High-throughput purification of combinatorial libraries II: Automated separation of single diastereomers from a 4-amido-pyrrolidone library containing intentional diastereomer pairs.

A 4-amido-pyrrolidone library that was intentionally synthesized as pairs of diastereomers was produced by solution-phase parallel syntheses and purified by an automated high-throughput purification system. A total of 2592 4-amido-pyrrolidinones were ultimately isolated as single diastereomers from a matrix of 1920 syntheses. After the four-step synthesis and HPLC purification, the average yield of a single diastereomer was 36.6%. The average chemical purity was >90%, and the average diastereomeric purity was >87%. The choice of chiral amines used to make amides with heterocyclic acid chlorides had a dramatic effect on success. Analysis of the relationship between amines used for synthesis and the diastereomeric separation showed that amides made from chiral 1,2-amino alcohols gave superior separation to amides from chiral morpholines. The presence of a hydrogen bond donor on the amide side chain seems to be required for a better diastereomeric separation.

Journal Article↗
PubMed2002

High-throughput screening in the diagnostics industry.

The diagnostics industry is constantly under pressure to bring innovation quicker to market and so the impetus to speed up product-development cycle times becomes greater. There are a number of steps in the product-development cycle where the application of high-throughput screening can help. In the case of lateral-flow immuno-diagnostics the selection of antibody reagents is paramount. In particular, rapid identification of antibody pairs that are able to 'sandwich' around the target antigen is required. One screen that has been applied successfully is the use of surface plasmon resonance biosensors like Biacore. Using such a system one can evaluate over 400 antibody pairings in under 5 days. Conventional approaches to screen this number of antibody pairs would take many months. Other automated screening systems like DELFIA can be used in processing the vast amount of tests required for clinical trials. In addition, the use of robotics to automate routine product testing can be used to shorten the product-development cycle.

Automation↗
PubMed2004

High-throughput production of Pyrococcus furiosus proteins: considerations for metalloproteins.

Free-living prokaryotic organisms contain all of the proteins required for the basic biochemical processes of life. As part of the Southeastern Collaboratory for Structural Genomics (SECSG), Pyrococcus furiosus is being used as a model system for developing a high-throughput protein expression and purification protocol. Its 1.9 million basepair genome encodes approximately 2200 putative proteins, less than 25% of which show similarity to any structurally characterized protein in the Protein Data Bank. The overall goal of the structural genomics initiative is to determine, in total, all existing protein folds. The immediate objective of this work is to obtain recombinant forms of all P. furiosus proteins in their functional states for structural determination. Proteins successfully produced by overexpression in another organism such as the bacterium Escherichia coli typically contain a single subunit, are soluble and do not contain (complex) cofactors. Analyses of the P. furiosus genome suggest that perhaps only a quarter of the genes encode proteins that would fall into this category. The hypothesis is that lack of the appropriate cofactor or of the partner protein(s) necessary to form a complex are major reasons why many recombinant proteins are insoluble. This work describes development of the production pipeline with attention to prediction and incorporation of cofactors.

Cloning, Molecular↗
PubMed2003

Virus-based expression systems facilitate rapid target in vivo functionality validation and high-throughput screening.

Target validation is one of rate-limiting steps in the modern drug discovery. The authors developed a strategy of combining adenovirus-mediated gene transfer for efficient target functionality validation, both in vivo and in vitro, with baculovirus expression to produce sufficient quantities of protein for high-throughput screening (HTS). The incorporation of green fluorescent protein (GFP) in the adenovirus vectors accelerates recombinant adenovirus plaque purification, whereas the use of epitope and affinity tags facilitates the identification and purification of recombinant protein. In this generalized scheme, the flexible modular design of viral vectors facilitates the transition between target validation and HTS. In the example presented, functional target validation in vivo was achieved by overexpressing the target gene in cell-based models and in the mouse cortex following adenovirus-mediated gene delivery. In this context, target overexpression resulted in the accumulation of a disease-related biomarker both in vitro and in vivo. A baculovirus-based expressional system was then generated to produce enough target protein for HTS. Thus, the use of these viral expression systems represents a generalized method for rapid target functionality validation and HTS assay development, which could be applied to numerous target candidates being elucidated in gene discovery programs.

Adenoviridae↗
PubMed2004

An investigation of liquid carryover and sample residual for a high-throughput flow cytometer sample delivery system.

The phenomenon of intersample contamination in air-segmented continuous-flow assays has been studied for many years, and new uses are being found for these sampling techniques every day. One application that has been developed recently employs a flow cytometer to conduct high-throughput screening assays of biological compounds. We have explored the sources of intersample contamination in the system and shown how methods developed previously can be applied to describe these phenomena. Using a simple model, we were able to accurately measure liquid film thickness in the sample tubing and demonstrate the effects of intersample contamination in a flow cytometer assay. Also, measures have been taken to reduce the level of intersample contamination in cytometric screening assays, helping to make the system a more viable tool for drug screening applications.

Capillaries↗
PubMedDate not supplied

Enhancing TOF/TOF-based de novo sequencing capability for high throughput protein identification with amino acid-coded mass tagging.

Because of the intrinsic physical properties of single- or double-charged ions, MALDI-based CID on these peptide precursor ions tends to be incomplete, resulting in a large number of MS/MS spectra unassigned or ambiguously identified. Consequently, the TOF/TOF high throughput capability may not be fully explored and utilized. Here, we describe a novel method for de novo sequence assignment of those MALDI TOF/TOF MS/MS spectra with incomplete or weak fragment ion series. In this approach, the deuterium-labeled lysine and leucine precursors were used in parallel to mass-tag the proteome of a metastatic human hepatocellular carcinoma (HCC) cell line during in vivo cell culturing. These stable isotope precursor markers not only position at terminal but at internal MS/MS fragment ions with the characteristic isotope pattern induced by multiple mass tagging in parallel. This enhanced signal specificity evidently resolved ambiguities in those sparse poor-quality TOF/TOF spectra by providing critical sequential links among MS/MS fragment ions. Our data-dependent approach was able to reduce many false-positives in current genome sequence-based peptide sequencing. With developing new algorithms accordingly, our approach is amenable for automation that will lead to more comprehensive and reliable identification for proteomes.

Carcinoma, Hepatocellular↗
PubMedDate not supplied

Serial isoelectric focusing as an effective and economic way to obtain maximal resolution and high-throughput in 2D-based comparative proteomics of scarce samples: proof-of-principle.

In 2D-based comparative proteomics of scarce samples, such as limited patient material, established methods for prefractionation and subsequent use of different narrow range IPG strips to increase overall resolution are difficult to apply. Also, a high number of samples, a prerequisite for drawing meaningful conclusions when pathological and control samples are considered, will increase the associated amount of work almost exponentially. Here, we introduce a novel, effective, and economic method designed to obtain maximum 2D resolution while maintaining the high throughput necessary to perform large-scale comparative proteomics studies. The method is based on connecting different IPG strips serially head-to-tail so that a complete line of different IPG strips with sequential pH regions can be focused in the same experiment. We show that when 3 IPG strips (covering together the pH range of 3-11) are connected head-to-tail an optimal resolution is achieved along the whole pH range. Sample consumption, time required, and associated costs are reduced by almost 70%, and the workload is reduced significantly.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2006

A semi-automated high-throughput approach to the generation of transposon insertion mutants in the nematode Caenorhabditis elegans.

The generation of a large collection of defined transposon insertion mutants is of general interest to the Caenorhabditis elegans research community and has been supported by the European Union. We describe here a semi-automated high-throughput method for mutant production and screening, using the heterologous transposon Mos1. The procedure allows routine culture of several thousand independent nematode strains in parallel for multiple generations before stereotyped molecular analyses. Using this method, we have already generated >17 500 individual strains carrying Mos1 insertions. It could be easily adapted to forward and reverse genetic screens and may influence researchers faced with making a choice of model organism.

Animals↗
PubMed2006

A high throughput method for genome-wide analysis of retroviral integration.

Retroviral and lentiviral vectors integrate their DNA into the host cell genome leading to stable transgene expression. Integration preferentially occurs in the proximity of active genes, and may in some case disturb their activity, with adverse toxic consequences. To efficiently analyze high numbers of lentiviral insertion sites in the DNA of transduced cells, we developed an improved high-throughput method called vector integration tag analysis (VITA). VITA is based on the identification of Genomic Tags associated to the insertion sites, which are used as signatures of the integration events. We use the capacity of MmeI to cleave DNA at a defined distance of its recognition site, in order to generate 21 bp long tags from libraries of junction fragments between vector and cellular DNA. The length of the tags is sufficient in most cases, to identify without ambiguity an unique position in the human genome. Concatenation, cloning and sequencing of the tags allow to obtain information about 20-25 insertion sites in a single sequencing reaction. As a validation of this method, we have characterized 1349 different lentiviral vector insertion sites in transduced HeLa cells, from only 487 sequencing reactions, with a background of <2% false positive tags.

Cell Line, Tumor↗
PubMed2003

Improvement of H2O2 stability of manganese peroxidase by combinatorial mutagenesis and high-throughput screening using in vitro expression with protein disulfide isomerase.

A functional expression system for a heme protein of Phanerochaete chrysosporium, manganese peroxidase (MnP), was developed using the Escherichia coli in vitro coupled transcription/translation system in the presence of hemin and fungal protein disulfide isomerase. This system has allowed the high-throughput construction and screening of a large diversity of mutant heme enzymes and has made it possible to improve the enzymatic function efficiently. Here we increased the H2O2 stability of MnP using this system; a mutant MnP library containing three randomized amino acid residues located in the H2O2-binding pocket of MnP was designed and constructed on a 384-well plate using SIMPLEX (single-molecule-PCR-linked in vitro expression). The screening of 10(4) samples independently expressed for improved H2O2 stability led to four positive mutants, the H2O2 stability of which was nine times higher than that of the wild-type.

Amino Acids↗
PubMed2005

Assays for membrane tyrosine kinase receptors: methods for high-throughput screening and utility for diagnostics.

The development of novel antagonists or agonists of membrane tyrosine kinase receptors is a large focus of discovery research. This review will provide some background on membrane tyrosine kinases as well as a description of some of the better established assays used for the high-throughput screening of membrane tyrosine kinase inhibitors. Biochemical methods detailed include those using labels such as radioactivity and fluorescence (fluorescence energy transfer, fluorescence and fluorescence polarization) as well as label-free assays using luminescence. These assays are solid phase, liquid phase, as well as bead based. In addition, a discussion on which tools are available to screen for membrane tyrosine kinase receptor modulators/activators using whole-cell assays will be presented. The potential clinical need for testing receptor activation/phosphorylation as well as the possibility of using some of these tests to measure biomarkers of disease or as clinical diagnostic tools to tailor drug therapy or monitor its efficacy will also be discussed.

Animals↗
PubMed2003

High-throughput functional affinity purification of mannose binding proteins from Oryza sativa.

We have used affinity chromatography in combination with mass spectrometry to isolate, identify, and assign a preliminary functional annotation to a large number of both known and novel proteins from rice. Rice (Oryza sativa) leaf, root, and seed tissue extracts were fractionated by column affinity chromatography using alpha-D-mannose as the ligand. Bound fractions were eluted and subjected to one-dimensional electrophoresis, followed by high-performance liquid chromatography-tandem mass spectrometric analysis of separated proteins. This multiplexed technology resulted in the isolation and identification of 136 distinct mannose binding proteins from rice. A comparative analysis demonstrates very little overlap of identified proteins between the respective tissues, and confirms the correctly compartmentalized presence of a significant number of proteins from largely tissue-specific biochemical pathways. Over 30% of the identified proteins with a previously annotated function are directly involved in sugar metabolism, including several highly expressed known rice lectins. Direct comparison of the peptide sequences identified in this study to those peptides identified in the most comprehensive survey of the rice proteome to date indicates that our current data represents a significant enrichment of proteins unique to this dataset. Nearly 15% of the identified proteins, identified on the basis of exact peptide matching to sequences in the rice genomic database, represent proteins without a previously known functional annotation, indicating the potential of this combined chromatographic approach to assign a preliminary function to novel proteins in a high-throughput fashion.

Binding, Competitive↗